COMPOSITIONS AND METHODS FOR TREATING CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/348,355, filed June 2, 2022 and U.S. Provisional Application Serial No. 63/433,157, filed December 16, 2022, the entire content of each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Most patients with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.

[0003] Recent developments using chimeric antigen receptor (CAR) modified autologous T cell therapy, which relies on redirecting genetically engineered T cells to a suitable cell-surface molecule on cancer cells, show promising results in harnessing the power of the immune system to treat B cell malignancies (see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). The clinical results with CD-19-specific CAR T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g., Kalos et al., Sci Transl Med 3 :95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368: 1509-1518 (2013)). An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma. [0004] Besides the ability of genetically modified T cells expressing a CAR or a second TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells requires the T cells to be capable of strong activation, expansion, persistence over time, and, in case of relapsing disease, to enable a ‘memory’ response. High and manageable clinical efficacy of CAR T cells is currently limited to BCMA- and CD- 19-positive B cell malignancies and to NY-ESO-1 -peptide expressing synovial sarcoma patients expressing HLA-A2. There is a clear need to improve genetically engineered T cells to more broadly act against various human malignancies.

[0005] Cytokine-associated toxicity such as CRS and neurotoxicity have been observed with CAR T- cell therapies (Bonifant et ah, Mol Ther Oncolytics. 2016;3: 16011; Turtle et ah, J Clin Invest. 2016; 126(6):2123-38). For example, existing CAR T-cell therapies are associated with severe CAR T-cell- related toxi cities, including cytokine release syndrome (CRS) and neurological events (NEs), that may limit administration to specialized treatment center (Yescarta Risk Evaluation and Mitigation Strategy (REMS) Gilead Pharma September 10, 2019; Kymriah Risk Evaluation and Mitigation Strategy (REMS) Novartis September 10, 2019) and impact use in difficult-to-treat patients.

[0006] In addition, while adoptive T cell therapies such as genetically modified T cells expressing a CAR or a second TCR have shown positive response rates in some cancer patients, tumor-induced immunosuppression remains a driver of resistance to this type of therapy (Leen et al., Annu Rev Immunol 25 (2007). Inhibitory receptors upregulated on activated T cells and their respective ligands expressed within the tumor milieu have been shown to contribute to T cell therapy failure (Abate - Daga et al., Blood 122(8) (2013). Among the inhibitory receptors, the programmed death receptor-1 (PD-1) plays a central role, given that recent studies have identified PD-1 expressed on tumor-antigen- specific T cells in tumors (Gros et al., J Clin Invest (2014)). The interaction of PD-1 with its ligand PD-L1 suppresses TCR signaling and T cell activation and thus prevents effective activation upon target recognition (Gros et al., J Clin Invest (2014); Yokosuka et al., J Exp Med 209(6) (2012); Ding et al., Cancer Res (2014); Karyampudi et al., Cancer Res (2014)). The clinical weight of these mechanisms is underlined by therapeutic studies combining adoptive cell therapy or gene-modified T cells with antibody -based PD-1 blockade that result in a marked improvement of anti -tumor activity (John et al., Clin Cancer Res 19(20) (2013); Goding et al., J Immunol 190(9) (2013). The systemic application of PD-1- or PD-L1 -blocking antibodies have the disadvantage of potentially targeting T cells of any reactivity and thus of inducing systemic side effects (Topalian et al., N Engl J Med 366(26) (2012); Brahmer et al, N Engl J Med 366(26) (2012)).

[0007] In view of the PD-L1 -mediated T cell inhibition, there is still a need to provide improved means having the potential to improve safety and efficacy of adoptive cell therapies and overcome the above disadvantages. There is further a need for adoptive cell therapies with a favorable benefit/risk profile, that are better tolerated by patients. Described herein are engineered T cells comprising PD-1 fusion proteins and modified T cell receptors, and methods of treatment comprising the use thereof, that address these and other needs. SUMMARY

[0008] Provided herein are adoptive cell therapies for treating a cancer that are useful to overcome problems associated with existing CAR T-cell therapies. Provided herein are improved methods for treating mesothelin (MSLN)-expressing cancers in human subjects.

[0009] Provided herein is a method for the treatment of a mesothelin (MSLN)-expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a composition comprising transduced T cells comprising:

(a) an anti-MSLN T cell receptor fusion protein (TFP), wherein the TFP comprises i. a T cell receptor (TCR) subunit comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, and ii. an antibody domain comprising an anti-MSLN antigen binding domain; and

(b) a fusion protein having a PD-1 polypeptide which is operably linked via its C-terminus to the N-terminus of an intracellular signaling domain of a costimulatory domain; wherein the composition is administered to the human subject in a dose of about 25 x 10 ⁶ to about 200 x 10 ⁶ transduced T cells.

[0010] In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 25 x 10 ⁶ transduced T cells. In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 50 x 10 ⁶ transduced T cells. In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 100 x 10 ⁶ transduced T cells. In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 130 x 10 ⁶ transduced T cells. In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 160 x 10 ⁶ transduced T cells. In some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose or amount of about 200 x 10 ⁶ transduced T cells.

[0011] In some embodiments, the method comprising administering the transduced T cells to the subject in need thereof further comprises administering to the human subject one or more additional doses of the transduced T cells. In some embodiments, the method comprises administering a second dose of the transduced T cells no sooner than about 4 months and no later than about 1 year following completion of a first dose.

[0012] In some embodiments, the method comprises administering the dose of the transduced T cells to the subject in a fractionated dosing regimen. In some embodiments, the dose is administered in two or more portions. For example, in some embodiments, a first portion of the dose is administered on Day 0 and a second dose is administered about 7 - 10 days later. For example, in some embodiments, a first portion of the dose is administered on Day 0 and a second dose is administered on or around Day 7. In some embodiments, the first portion is about 33% of the dose (e.g., about 33% of 25 x 10 ⁶ transduced cells, about 33% of 50 x 10 ⁶ transduced cells, about 33% of 100 x 10 ⁶ transduced cells, about 33% of 130 x 10 ⁶ transduced cells, about 33% of 160 x 10 ⁶ transduced cells, or about 33% of 200 x 10 ⁶ transduced cells) and the second portion of the dose is about 67% of the dose (e.g., about 67% of 25 x 10 ⁶ transduced cells, about 67% of 50 x 10 ⁶ transduced cells, about 67% of 100 x 10 ⁶ transduced cells, about 67% of 130 x 10 ⁶ transduced cells, about 67% of 160 x 10 ⁶ transduced cells, or about 67% of 200 x 10 ⁶ transduced cells). In some embodiments, the first portion is about 50% of the dose e.g., about 50% of 25 x 10 ⁶ transduced cells, about 50% of 50 x 10 ⁶ transduced cells, about 50% of 100 x 10 ⁶ transduced cells, about 50% of 130 x 10 ⁶ transduced cells, about 50% of 160 x 10 ⁶ transduced cells, or about 50% of 200 x 10 ⁶ transduced cells) and the second portion of the dose is about 50% of the dose e.g., about 50% of 25 x 10 ⁶ transduced cells, about 50% of 50 x 10 ⁶ transduced cells, about 50% of 100 x 10 ⁶ transduced cells, about 50% of 130 x 10 ⁶ transduced cells, about 50% of 160 x 10 ⁶ transduced cells, or about 50% of 200 x 10 ⁶ transduced cells).

[0013] In some embodiments, the method comprises determining PD-L1 expression on biopsy tissue at baseline, prior to administration of the transduced T cells to the subject. In some embodiments, a dosing level is determined according to the baseline PD-L1 level. In some embodiments, the method comprising administering the transduced T cells to the subject in need thereof comprises administering the transduced T cells to the subject via intravenous infusion. In some embodiments, the transduced T cells are administered as a single agent.

[0014] In some embodiments, a dose range of ± 15% of a target dose is administered. For example, in some embodiments, the composition comprising the transduced T cells is administered to the human subject in a dose range of ± 15% of a target dose, wherein the target dose is about 25 x 10 ⁶ , about 50 x 10 ⁶ , about 100 x 10 ⁶ , about 130 x 10 ⁶ , about 160 x 10 ⁶ , or about 200 x 10 ⁶ transduced T cells.

[0015] In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is an unresectable, metastatic, or recurrent cancer. In some embodiments, the human subject has been diagnosed with the cancer. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is malignant pleural mesothelioma (MPM). In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is serous ovarian adenocarcinoma. In some embodiments, the serous ovarian adenocarcinoma is serous ovarian, fallopian tube, or primary peritoneal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, cancer is triple negative breast cancer. In some embodiments, the subject has received at least 1 systemic therapy for metastatic or unresectable disease prior to administration of the transduced T cells. In some embodiments, the subject has received 1, 2, 3, 4, or 5 systemic therapies for metastatic or unresectable disease prior to administration of the transduced T cells.

[0016] In some embodiments, the method comprising administering the transduced T cells to the subject in need thereof further comprises administering to the human subject a lymphodepleting chemotherapy regimen prior to administration of the transduced T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of about four doses of fludarabine and about three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of four doses of fludarabine and three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of about 30 mg/m ² /day on days -7 through -4 relative to administration of the transduced T cells, and further comprises administration of cyclophosphamide at a level of about 600 mg/m ² /day on days -6 through -4 relative to administration of the transduced T cells; wherein the transduced T cells are administered on day 0. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 30 mg/m ² /day on days -7 through -4 relative to administration of the transduced T cells, and further comprises administration of cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -4 relative to administration of the transduced T cells; wherein the transduced T cells are administered on day 0.

[0017] In some embodiments, the method further comprises retreatment of the human subject after completion of an initial administration of the composition, wherein the retreatment comprises administration of a lymphodepleting chemotherapy regimen followed by a second dose of the composition comprising the transduced T cells. In some embodiments, certain patient populations are eligible for retreatment. For example, in some embodiments, the human subject exhibited a confirmed response (a partial response (PR) or complete response (CR) or at least stable disease (SD) for more than 4 months following the initial administration of the composition, and subsequently exhibited relapse of disease. As another example, in some embodiments, the human subject (i) exhibited an objective response (PR or CR) after the initial administration of the composition and developed signs and symptoms of progression, and/or (ii) exhibited a best response of stable disease (SD) , sustained for at least 8 weeks following the initial administration of the composition. Thus, in some embodiments, the method provided herein comprises administration of a first dose of the transduced T cells, followed by retreatment comprising a second dose of the transduced T cells in certain patient populations. In some embodiments, the retreatment comprises a reduced lymphodepletion chemotherapy regimen. In some embodiments, the retreatment comprises administration of lymphodepleting chemotherapy comprising administration of fludarabine at a level of 30 mg/m ² /day on days -7 through -5 relative to administration of the transduced T cells, and further comprises administration of cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -5 relative to administration of the transduced T cells. In some embodiments, if bendamustine is used as a part of the retreatment regimen, the regimen can be abbreviated to 70 mg/m ² /day IV on days -5 and -4 (e.g., 2 doses of bendamustine). In some embodiments, the retreatment is no sooner than 4 months, no sooner than 6 months, no sooner than 8 months, or no sooner than 10 months following completion of the initial administration of the composition. In some embodiments, the retreatment is no later than 1 year following completion of the initial administration of the composition.

[0018] In an aspect, the present disclosure provides a method for the treatment of a mesothelin (MSLN)-expressing cancer in a human subject in need thereof, the method comprising:

(a) administering to the human subject a lymphodepleting chemotherapy regimen; and

(b) administering to the human subject a composition comprising transduced T cells comprising i. an anti-MSLN T cell receptor fusion protein (TFP), wherein the TFP comprises

A. a T cell receptor (TCR) subunit comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, and

B. an antibody domain comprising an anti-MSLN antigen binding domain; and ii. a fusion protein having a PD-1 polypeptide which is operatively linked via its C-terminus to the N-terminus of an intracellular signaling domain of a costimulatory domain; wherein the transduced T cells are administered in a target dose of about 50 x 10 ⁶ , about 100 x 10 ⁶ , about 130 x 10 ⁶ , about 160 x 10 ⁶ , or about 200 x 10 ⁶ transduced T cells.

[0019] In some embodiments, a dose range of ± 15% of the target dose is administered. In some embodiments, a second dose of the transduced T cells is administered no sooner than about 4 months following administration of a first dose of the transduced T cells and no later than about 12 months following administration of the first dose of the transduced T cells. In some embodiments, the composition is administered via intravenous infusion. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of four doses of fludarabine and three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 30 mg/m ² /day on days -7 through -4 relative to administration of the transduced T cells, and further comprises administration of cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -4 relative to administration of the transduced T cells, wherein the transduced T cells are administered on day 0.

[0020] In some embodiments, prior to administration of the transduced T cells, 50% or more of tumor cells of the subject express MSLN with 1+, 2+, and/or 3+ intensity as measured by immunohi stochemi stry .

[0021] In some embodiments, the method comprising administering the transduced T cells to the subject in need thereof further comprises determining the expression of MSLN on tumor cells of the subject prior to administration of the transduced T cells, wherein 50% or more of tumor cells of the subject express MSLN with 1+, 2+, and/or 3+ intensity as measured by immunohistochemistry. In some embodiments, the cancer is epithelioid MPM, and no confirmation of MSLN expression may be required or conducted prior to administration of the transduced T cells. For example, in some embodiments, the transduced T cells are administered to epithelioid MPM patients having any MSLN expression level or intensity.

[0022] In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is an unresectable, metastatic, or recurrent cancer. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is selected from the group consisting of malignant pleural mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, and triple negative breast cancer. In some embodiments, the serous ovarian adenocarcinoma is serous ovarian, fallopian tube, or primary peritoneal cancer. In some embodiments, the subject has received at least 1 systemic therapy for metastatic or unresectable disease prior to administration of the transduced T cells. In some embodiments, the subject has received 1, 2, 3, 4, or 5 systemic therapies for metastatic or unresectable disease prior to administration of the transduced T cells.

[0023] In an aspect, the present disclosure provides a method of treating a MSLN-expressing cancer in a human subject, the method comprising

(a) determining that 50% or more of tumor cells of the subject express MSLN as measured by immunohistochemistry; and

(b) administering to the subject at least one dose of transduced T cells comprising i. an anti-MSLN T cell receptor fusion protein (TFP), wherein the TFP comprises

A. a T cell receptor (TCR) subunit comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain, and

B. an antibody domain comprising an anti-MSLN antigen binding domain; and ii. a fusion protein having a PD-1 polypeptide which is operatively linked via its C-terminus to the N-terminus of an intracellular signaling domain of a costimulatory domain; wherein the transduced T cells are administered in a target dose of about 50 x 10 ⁶ , about 100 x 10 ⁶ , about 130 x 10 ⁶ , about 160 x 10 ⁶ , or about 200 x 10 ⁶ transduced T cells.

[0024] In some embodiments, the intensity of MSLN expression is 1+, 2+, and/or 3+ in 50% or more of tumor cells of the subject.

[0025] In some embodiments, the methods provided herein further comprise a leukapheresis step prior to administration of the transduced T cells. In some embodiments, the leukapheresis step comprises collection of peripheral blood mononuclear cells (PBMCs) from human subject. In some embodiments, the leukapheresis is performed prior to administration of a lymphodepleting chemotherapy regimen. [0026] In some embodiments, the methods provided herein do not induce cytokine release syndrome (CRS) in the subject above grade 1, above grade 2, or above grade 3.

[0027] In some embodiments, the present disclosure provides methods for treating a mesothelin (MSLN)-expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a composition comprising transduced T cells provided herein comprising a TFP and a fusion protein having a PD-1 polypeptide, wherein the PD-1 polypeptide comprises the extracellular domain of PD-1 or a portion thereof. In some embodiments, the PD-1 polypeptide comprises the extracellular domain of PD-1 or a portion thereof that is capable of interacting with a ligand for PD-1 (e.g., PDL1 or PDL2). In some embodiments, the PD-1 polypeptide comprises an extracellular domain of PD-1 or a portion thereof, and further comprises the transmembrane domain of PD-1. In some embodiments, the PD-1 polypeptide comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 56. In some embodiments, the PD-1 polypeptide comprises the amino acid sequence of SEQ ID NO: 56.

[0028] In some embodiments, the present disclosure provides methods for treating a mesothelin (MSLN)-expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a composition comprising transduced T cells, wherein the transduced T cells comprise a TFP and a fusion protein having a PD-1 polypeptide and an intracellular signaling domain of a costimulatory domain. In some embodiments, the costimulatory domain is CD28. In some embodiments, the intracellular signaling domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 57. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the fusion protein having the PD-1 polypeptide comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 58. In some embodiments, the fusion protein having the PD-1 polypeptide comprises the amino acid sequence of SEQ ID NO: 58.

[0029] In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0030] In some embodiments, the TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0031] In some embodiments, the TCR intracellular domain comprises an intracellular domain of TCR alpha, TCR beta, TCR delta, or TCR gamma, or an amino acid sequence having at least one modification thereto.

[0032] In some embodiments, the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto. In some embodiments, the intracellular signaling domain is CD3 epsilon.

[0033] In some embodiments, at least two or three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from a same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. In some embodiments, at least of two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. In some embodiments, all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, the TCR subunit comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the TCR subunit comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the TCR subunit comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the TFP comprises the amino acid sequence of SEQ ID NO: 52. [0034] In some embodiments, the TCR subunit and the anti-MSLN antigen binding domain are operatively linked.

[0035] In some embodiments, the TFP functionally interacts with an endogenous TCR complex in the T cell. In some embodiments, the TFP incorporates into a TCR complex in the T cell.

[0036] In some embodiments, the antibody domain is a murine, human or humanized antibody domain. In some embodiments, the anti-MSLN binding domain is a scFv or a VHH domain. In some embodiments, the anti-MSLN binding domain comprises a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 47. In some embodiments, the anti-MSLN binding domain is a VHH domain. In some embodiments, the anti-MSLN binding domain comprises the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 47. In some embodiments, the antibody domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker sequence is 120 amino acids in length or less. In some embodiment, the linker sequence comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In further embodiments, n is an integer from 1 to 4. In some embodiments, the linker sequence is a G4S linker with additional amino acids at the N- and/or the C-terminus of the linker. For example, in some embodiments, the linker is a G4S sequence with 1, 2, 3, 4, or 5 amino acids at the N-terminus (N-terminal to the first Gly of the G4S sequence) and/or with 1, 2, 3, 4, or 5 amino acids at the C-terminus (C-terminal to the last Ser of the G4S sequence). In some embodiments, the linker sequence comprises an amino acid sequence according to SEQ ID NO: 61. In some embodiments, the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are each from the same TCR subunit, wherein the TCR subunit is CD3 epsilon.

[0037] In some embodiments, the transduced T cells are human T cells. In some embodiments, the transduced T cells are CD8+ T cells and/or CD4+ T cells. In some embodiments, the transduced cells are alpha beta T cells. In some embodiments, the transduced T cells are autologous or allogeneic T cells.

[0038] In some embodiments, the method further comprises obtaining a population of cells from the human subject prior to administering transduced T cells, and transducing T cells from the population of cells with a recombinant nucleic acid comprising a sequence encoding the TFP and a sequence encoding the fusion protein having a PD-1 polypeptide, thereby generating the transduced T cells.

[0039] In some embodiments, the human subject has or has not received a lymphodepleting chemotherapy regimen prior to administering the first portion of a dose.

[0040] In some embodiments, the human subject is at risk of recurrence. In some embodiments, the human subj ect has a prior history of recurrence after a prior therapy. In some embodiments, the MSLN- expressing cancer is locally advanced. In some embodiments, the MSLN-expressing cancer is metastatic. In some embodiments, the MSLN-expressing cancer is unresectable.

[0041] In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 25 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 50 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 100 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 130 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 160 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is MPM, wherein the dose is about 200 x 10 ⁶ transduced cells.

[0042] In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 25 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 50 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 100 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 130 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 160 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is serous ovarian adenocarcinoma, wherein the dose is about 200 x 10 ⁶ transduced cells.

[0043] In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 25 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 50 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 100 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 130 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 160 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma, wherein the dose is about 200 x 10 ⁶ transduced cells.

[0044] In some embodiments, the MSLN-expressing cancer is triple negative breast cancer, wherein the dose is about 25 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is triple negative breast cancer, wherein the dose is about 50 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is triple negative breast cancer, wherein the dose is about 100 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is triple negative breast cancer, wherein the dose is about 130 x 10 ⁶ transduced cells. In some embodiments, the MSLN- expressing cancer is triple negative breast cancer, wherein the dose is about 160 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is triple negative breast cancer, wherein the dose is about 200 x 10 ⁶ transduced cells.

[0045] In some embodiments, the MSLN-expressing cancer is colorectal cancer, wherein the dose is about 25 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is colorectal cancer, wherein the dose is about 50 x 10 ⁶ transduced cells. In some embodiments, the MSLN- expressing cancer is colorectal cancer, wherein the dose is about 100 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is colorectal cancer, wherein the dose is about 130 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is colorectal cancer, wherein the dose is about 160 x 10 ⁶ transduced cells. In some embodiments, the MSLN-expressing cancer is colorectal cancer, wherein the dose is about 200 x 10 ⁶ transduced cells.

INCORPORATION BY REFERENCE

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1A and FIG. IB are schematic representations of the TC-510 bicistronic transgene (FIG. 1A) and the TC-510 T cell receptor fusion construct interacting with a tumor cell (FIG. IB).

[0048] FIG. 2A shows the tumor volume (mm ³ ) over time in NSG mice injected with MSTO-MSLN- PDLl-Luc tumor cells, and treated with NT control cells, Vehicle, or TC-510 at the indicated doses. Average tumor volume in each group is shown. FIG. 2B provides the individual tumor volumes in each treatment group. N= 8 per group. TC-510 groups treated with 1.5xl0 ⁶ and LOxlO ⁶ TC-510 cells exhibited dramatic tumor regression, with nearly complete tumor regression by day 24 (7/8 mice) and complete tumor regression in 8/8 mice was by day 28. The TC-510 0.5xl0 ⁶ treatment group also had complete tumor regression (8/8 mice) by day 28 (p=0.0001, Two Way ANOVA, Dunnet’s multiple comparison) compared to NT treatment. Mice in the lowest treatment group, 0. IxlO ⁶ , had 3 mice with tumors 90-140 mm ³ and 5 mice with tumors 800-2100 mm ³ and no mice with complete tumor regression on Day 28.

[0049] FIGs. 3A-3C show the body weight over time of NSG mice injected with MISTO-MSLN- PDLl-Luc tumor cells, and treated with NT control cells, Vehicle, or TC-510 at the indicated doses. FIG. 3A shows the mean body weights in each group (+/- SD). FIG. 3B shows the individual body weights in each group. FIG. 3C shows the percent weight change for each treatment group. [0050] FIG. 4 is a schematic representation of the clinical study described in Example 2.

[0051] FIG. 5A shows schematic representations of the TC-210 construct and the TC-210 bicistronic constructs containing sequences encoding the PD-lxCD28 switch receptor with the transmembrane region of either PD1 (PD1TM) or CD28 (CD28TM). FIG. 5B shows flow cytometry plots depicting co-expression of PD-1 and the TC-210 T cell receptor fusion construct (TRuC) at day 10 of expansion. FIG. 5C shows transduction efficiency of the T cell receptor fusion construct, measured as percentage of CD3 ⁺ cells, across NT control, TC-210, PD1TM, and CD28TM groups. The CD28TM group showed significantly lower transduction compared to the PD1TM group. FIG. 5D shows median florescence intensity (MFI) of TRuC receptor expression of TRuC+ T cells. Both the PD1TM and CD28TM groups showed statistically minor reductions in expression levels compared to TC-210 alone. FIG. 5E shows MFI of PD-1 expression of TRuC ⁺ T cells. Both the PD1TM and CD28TM groups displayed similar levels of PD-1 expression. FIG. 5F shows the frequency of CD8+ T cells (e.g., the ratio of CD4 ⁺ to CD8 ⁺ T cells) of TRuC ⁺ T cells on day 10 of process. All transduced groups displayed a significantly increased ratio compared to the NT group.

[0052] FIG. 6A shows tumor cell cytotoxicity of MSLN TRuC T cells after co-cultured with MSLN- negative (C30), MSTO-MSLN, and MSTO-MSLN-PDL1 cell lines. Equivalent levels in tumor lysis were seen for all three TRuC T cell products against both MSTO-MSLN and MSTO-MSLN-PDL1. FIG. 6B shows levels of cytokine secretion (IFN-y, IL-2, GM-CSF, and TNF-a) between TC-210 and the PD-lxCD28 switch receptor groups, measured from the culture supernatants of the cytotoxicity assay by MSD ELISA. FIG. 6C shows levels of cytokine production after 24 hours 1 : 1 co-culture of MSLN TRuC T cells with MSTO-MSLN, MSTO-MSLN-PDL1, or MSTO-MSLN-PDL1 cells in the presence of an anti -PD-1 monoclonal antibody (aPD-a). Blockade of the interaction between PD- lxCD28 switch receptor and PD-L1 reduced cytokine production in TRuC T cells with the switch receptor. FIG. 6D shows detection of phosphorylated ERK (pERK) following co-culture of TRuC T cells and MSTO, MSTO-MSLN, and MSTO-MSLN-PDL1 cells. FIG. 6E shows the percentage of pERK of TRuC+ cells after 10 minutes incubation. FIG. 6F shows the MFI of pERK+ TRuC+ cells at 10 minutes incubation. FIG. 6G shows the percentage of pERK of TRuC+ cells after 60 minutes incubation. FIG. 6H shows the MFI of pERK+ TRuC+ cells at 60 minutes of incubation. Plotted data represent 2-3 individual donors and are plotted as mean (± SEM). Data were analyzed for statistical significance by two-way ANOVA.

[0053] FIG. 7A shows levels of cytokine production, measured by MSD ELISA, from TRuC T cells cultured with MSLN and increasing concentrations of PD-Ll-Fc (pg/mL). MSLN antigen alone induced comparable levels of cytokine production by all TRuC T cells. Upon stimulation of MSLN and PD-L1, both PD-lxCD28 switch receptor groups (PD1TM and CD28TM) displayed increased levels of cytokines relative to TC-210. FIG. 7B shows levels of cytokine production from MSLN TRuC-T cells cultured at a 1 : 1 ratio with low-MSLN antigen expressing cell line C30, C30 overexpressing PD-L1 (C30-PDL1), parental MSTO, and MSTO over-expressing PD-L1 (MST0-PDL1). The CD28TM group showed a significantly increased cytokine response when cultured with the parental MSTO cell line.

[0054] FIG. 8A shows schematic representations of the TC-210 construct and TC-210 bicistronic constructs containing the anti-mesothelin TRuC followed by a sequence encoding PD1TM, or the PD1TM variants (PDlTM ^Mutant and PDl ^Trunc ) made non-functional through mutation or deletion of the CD28 signaling domain. FIG. 8B shows flow cytometry plots depicting PD-1 and TRuC receptor expression on TRuC T cells. Both PD-lxCD28 switch receptor variants were expressed and showed positive expression for TRuC and exogenous PD-1 receptor. FIG. 8C shows the MFI of pERK of TRuC+ T cells across the different TRuC T cell groups after culture at a 3: 1 effector-to-target ratio with MSLN-expressing cells (n=2). FIG. 8D shows levels of cytokine production from TRuC T cells plated at a 1 : 1 ratio with MSLN-expressing tumor cells for 72 hours. Supernatants collected from coculture with tumor cells and either variant (PDlTM ^Mutant or PDl ^Trunc ) yielded limited cytokine secretion relative to PD1TM.

[0055] FIG. 9A shows the fold expansion of TRuC T cells, normalized for transduction efficiency, following an in vitro rechallenge assay with MSTO ^MSLN ' ^PD ' ^L1 tumor cells at a 1 :20 effector-to-target ratio every 96 hours. The experimental groups included NT, TC-210, TC-510 (engineered TRuC T cells with anti-MSLN antigen binding domain and PD1TM domain associated with the PD-lxCD28 switch receptor), and the PDlTM ^Mutant and PDl ^Trunc variants. FIG. 9B shows the levels of cytokines across experimental groups from culture supernatants collected 72 hours after each antigen challenge. There were no discernible differences between TC-210, PDlTM ^Mutant , and PDl ^Trunc cultures in terms of cytokine production. Statistical analysis was carried out with a two-way ANOVA. FIG. 9C shows brightfield microscopy images of culture morphology across experimental groups on day 8 of culture. TC-510 cultures showed visible clustering compared to the more diffuse pattern of cells in the TC- 210, PDlTM ^Mutant , and PDl ^Trunc cultures. FIG. 9D shows FACs plots of CD3 and TRuC receptor expression on viable CD45 ⁺ from MSTO ^MSLN ' ^PD ' ^L1 cultures on day 12 of culture and flow cytometry plots depicting TIGIT and LAG-3 exhaustion marker expression by TRuC T cells.

[0056] FIG. 10A shows the tumor volume (mm ³ ) over time in NSG mice injected with 1 x 10 ⁶ MSTO- MSLN-PDLl-Luc tumor cells, and treated with NT control cells, Vehicle, TC-210, TC-510, or Naive. Engineered human T cells were administered at a dose of 2.0 x 10 ⁶ TRuC+ T cells per mouse, when the tumor size was between 150-200 mm ³ . Data are representative of two independent experiments with two donors (n=10 mice per group) and are shown as the average of the experiments. FIG. 10B provides the individual tumor volumes in the TC-210 and TC-510 treatment groups. N=10 per group. Both groups showed similar anti -tumor activity with complete tumor clearance by day 17. A tumor rechallenge was performed 44 days after T cell administration, without retreatment. Following the rechallenge, all the TC-210 treated mice experienced tumor recurrence, whereas recurrence was limited to 1/8 mice in the TC-510 group.

[0057] FIG. 11A shows memory-effector populations of CD8+ TRuC-T cells in NT, TC-210, PD1TM, and CD28TM groups following 10 days of expansion. FIG. 11B shows histograms of flow cytometric analysis of additional phenotypic markers of T cell activation and exhaustion. FIG. 11C shows expression of surface expression markers CCR7, CD25, CD28, CD45RA, CD69, CD70, LAG- 3, and TIM-3 represented as the percentage of TRuC+ cells and MFI of TRuC+ cells.

[0058] FIG. 12 shows endogenous levels of PD-L1 expression by the parental C30, Suit2, and MSTO tumor lines in co-culture with MSLN TRuC-T cells.

[0059] FIG. 13A shows levels of IFN-y (pg/mL) produced in response to increasing amounts of Fc- MSLN in TC-210 TRuC-T cells. FIG. 13B shows the fold expansion of TRuC T cells, after 96 hours, cultured under 1.0 pg/mL of MSLN with increasing concentrations of PD-Ll-Fc. FIG. 13C shows the percentage of viable CD3 ⁺ cells across TRuC T cell groups under 1.0 pg/mL of MSLN with increasing concentrations of PD-Ll-Fc.

DETAILED DESCRIPTION

[0060] Described herein are methods of adoptive cell therapy for treating a cancer, e.g., a mesothelin- expressing cancer, using T cell receptor fusion protein (TFP) molecules (also referred to herein as T cell receptor fusion construct or TRuC™) directed to mesothelin-expressing tumor cells. The limited efficacy of CARs in solid tumors is thought to be due to a number of factors including T cell exhaustion and lack of persistence. Chimeric antigen receptors are physically removed from the native TCR and signal only through one of the 6 TCR subunits (i.e., the CD3(^ chain). The failure to initiate and harness a complete TCR response is arguably a primary underlying factor preventing CAR T cell success in solid tumor indications. The present disclosure provides, inter alia, a T cell engineering platform based on a T cell receptor fusion construct (TFP or TRuC) including a tumor antigen binding domain. An engineered T cell product referred to herein as “TC-510” comprises T cells with a TFP/TRuC including an anti -MSLN antigen binding domain and a PD-lxCD28 switch, designed to bypass the limitations of CARs and other adoptive T cell therapies.

[0061] TC-510 comprises genetically engineered T cells that express a PD-lxCD28 switch receptor, and that further express a humanized single-domain antibody that recognizes human MSLN fused to the cluster of differentiation (CD) 3 a subunit which, upon expression, is incorporated into the endogenous TCR complex. There, it redirects the T cells to recognize and eliminate MSLN-positive tumor cells. Furthermore, the addition of the PD-lxCD28 switch receptor helps with overcoming PD- L1/PD-L2 mediated immunosuppression and turns this inhibitory function into a costimulatory signal. Thus, without wishing to be bound by theory, the PD-lxCD28 switch receptor performs a decoy function as well as providing a costimulation signal to enhance T cell activity upon interaction with a PD-L1 expressing tumor cell, such enhanced T cell activity including proliferation, cytokine production, and persistence.

[0062] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

Adoptive T cell Therapy

[0063] Adoptive T cell therapy (ACT) is a therapeutic modality that involves the manipulation of a cancer patient’s own T cells to endow them with anti -tumor activity. This is accomplished through the collection, ex vivo activation, modification and expansion, and re-infusion into the patient. The objective of the process is the generation of potent and cancer antigen-specific T cell immunity. Tumor-associated antigens can be classified into 3 major groups:

1. Antigens present in healthy tissue but over-expressed in tumors, usually because they confer a growth advantage to the cancer cell.

2. Neo-antigens arising from somatic mutations in cancer cells.

3. Cancer germline antigens, which are proteins expressed on germline cells, which reside in immunoprivileged sites, and therefore are not vulnerable to autoimmune T cell targeting.

[0064] The first successful application of ACT was the use of tumor infiltrating lymphocytes (TILs), which rendered clinical responses in approximately 50% of patients with malignant melanoma (Topalian et. al., 1988). The wide applicability of this therapeutic modality was hindered by the requisite surgery to procure tissue from which to isolate TILs, the difficulties in successfully isolating and expanding TILs, and the difficulty in reproducing similar results in other malignancies. Genetransfer-based strategies were developed to overcome the immune tolerance on the tumor-specific T cell repertoire. These approaches redirect T cells to effectively target tumor antigens through the transfer of affinity-optimized T cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs) via retrovirus- or lentivirus-based stable transduction. The CAR T cells represent the most extensively characterized ACT platform. CAR T cells are autologous T cells that have been re-programmed to target surface-expressed cancer associated antigens, typically through the inclusion of a single chain antibody variable fragment (scFv). These binding domains are fused to co-stimulatory domains as well as the CD3(^ chain and subsequently transfected into autologous T cells using viral or non-viral transduction processes. Upon binding to its cognate antigen, CAR T phosphorylates the immunoreceptor tyrosine-based activation motifs (IT AMs) within the CD3 zeta chain. This serves as the initiating T cell activation signal and is critical for CAR T mediated lysis of tumor antigens. Concurrently, scFv binding also stimulates the fused co-simulation domains (usually CD28 or 4-1BB) which provide important expansion and survival signals. Two CD19-directed CAR T cell approaches were approved in 2017 by FDA for the treatment of patients with either pediatric acute lymphoblastic leukemia (ALL) or diffuse large B-cell lymphoma (DLBCL), respectively: tisagenlecleucel (Kymriah™) and axicabtagene cileucel (Yescarta™) (CBER, 2017a; CBER 2017b). The former was also approved by FDA in 2018 for the treatment of patients with relapsed/refractory DLBCL. Notwithstanding this activity in hematological malignancies, CAR T cells have failed to induce significant clinical efficacy against solid cancers, largely due to T cell exhaustion and very limited persistence. By utilizing only 1 (CD3(^ chain) of the 6 distinct T cell receptor subunits in combination with a costimulatory domain, CARs operate outside of the natural TCR signaling complex. The failure to initiate and harness a complete TCR response is arguably a primary underlying factor preventing CAR T cell success in solid tumor indications.

TFP Technology

[0065] In some embodiments, the isolated TFP molecules comprise a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto. In some embodiments, the anti-mesothelin binding domain is connected to the TCR extracellular domain by a linker sequence. In some instances, the linker region comprises (G4S)n, wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.

[0066] In some embodiments, the isolated TFP molecules further comprise a sequence encoding a costimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.

[0067] Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), fusion proteins comprising PD-1 and a co-stimulatory domain, T cells engineered to express one or more TFPs and a fusion protein comprising PD-1 and a costimulatory domain, and methods of use thereof for the treatment of diseases. In one aspect, provided herein is an isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit and a human or humanized antibody domain; and encoding a fusion protein comprising a PD-1 polypeptide or fragment thereof and a costimulatory domain. An exemplary nucleic acid molecule is shown schematically in FIG. 1A.

[0068] Also provided herein are vectors that comprise a nucleic acid molecule encoding any of the TFP molecules and/or fusion proteins provided herein. In some embodiments, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3’UTR.

[0069] Also provided herein are cells that comprise any of the described vectors. In some embodiments, the cell is a human T cell. In some embodiments, the cell is a CD8+ and/or CD4+ T cell. In other embodiments, the cell is selected from a naive T-cell, memory stem T cell, central memory T cell, double negative T cell, effector memory T cell, effector T cell, ThO cell, TcO cell, Thl cell, Tel cell, Th2 cell, Tc2 cell, Thl7 cell, Th22 cell, gamma/delta T cell, alpha/beta T cell, natural killer (NK) cell, natural killer T (NKT) cell, hematopoietic stem cell and pluripotent stem cell. In some embodiments, the cell comprises a nucleic acid encoding a TFP provided herein, and further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD-1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.

[0070] In an aspect, the isolated TFP molecule provided herein that comprises a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.

[0071] In an aspect, the isolated TFP molecule provided herein that comprises a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, is capable of functionally integrating into an endogenous TCR complex.

[0072] In another aspect, provided herein are human CD8+ and/or CD4+ T cells that comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell. In embodiments, the CD8+ and/or CD4+ T cells further comprise a fusion protein comprising a first polypeptide comprising at least a portion of PD-1 and a second polypeptide comprising a costimulatory domain. In embodiments, the costimulatory domain is a CD28 domain. In embodiments, the fusion protein comprises a PD-1 extracellular domain, a PD-1 transmembrane domain, and a CD28 intracellular domain.

[0073] In another aspect, provided herein are protein complexes that comprise i) a TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex. In embodiments, the protein complexes further comprise and/or are expressed together with a fusion protein comprising a first polypeptide comprising at least a portion of PD-1 and a second polypeptide comprising a costimulatory domain (e.g., a PD-lxCD28 switch receptor).

[0074] In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-mesothelin binding domain is connected to the TCR extracellular domain by a linker sequence. In some instances, the linker region comprises (G4S)n, wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.

[0075] In another aspect, provided herein is a population of human CD8+ and/or CD4+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell; and wherein the T cells of the population further individually or collectively comprise at least one PD- lxCD28 switch receptor.

[0076] In another aspect, provided herein is a population of human CD8+ and/or CD4+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein and at least one PD-lxCD28 switch receptor encoded by an isolated nucleic acid molecule provided herein.

[0077] In another aspect, provided herein are methods of making a cell comprising transducing a T cell with any of the described vectors.

[0078] In another aspect, provided herein are methods of generating a population of RNA-engineered cells that comprise introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding any of the described TFP molecules and/or fusion proteins.

[0079] In another aspect, provided herein are methods of providing an anti-tumor immunity in a mammal that comprise administering to the mammal an effective amount of a cell expressing any of the described TFP molecules and any of the described PD-1 fusion proteins (e.g., PD-lxCD28 switch receptors). In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.

[0080] In another aspect, provided herein are methods of treating a mammal having a disease associated with expression of mesothelin that comprise administering to the mammal an effective amount of the cell comprising any of the described TFP molecules and/or fusion proteins. In some embodiments, the disease associated with mesothelin expression is selected from a proliferative disease such as a cancer or malignancy, or a precancerous condition. Examples include, but are not limited to, a pancreatic cancer, an ovarian cancer, a stomach cancer, a colorectal cancer, a lung cancer, cholangiocarcinoma, mesothelioma, a breast cancer, or an endometrial cancer; or a non-cancer related indication associated with expression of mesothelin. In some embodiments, the disease associated with mesothelin expression is a solid tumor. In some embodiments, the disease associated with mesothelin expression is a mesothelin-expressing recurrent, metastatic, or unresectable solid tumor. In some embodiments, the disease associated with mesothelin expression is malignant pleural/peritoneal mesothelioma (MPM), ovarian cancer, pancreatic cancer, colorectal cancer, or triple negative breast cancer. In some embodiments, the disease associated with mesothelin expression is metastatic or unresectable MPM, ovarian cancer, pancreatic cancer, colorectal cancer, or triple negative breast cancer. [0081] In some embodiments, the cells expressing any of the described TFP molecules and/or fusion proteins are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule and/or fusion protein. In some embodiments, the cells expressing any of the described TFP molecules and/or fusion proteins are administered in combination with an agent that treats the disease associated with mesothelin.

[0082] Also provided herein are any of the described isolated nucleic acid molecules, any of the described isolated polypeptide molecules, any of the described isolated TFPs, any of the described PD- 1 switch receptors, any of the described protein complexes, any of the described vectors or any of the described cells for use as a medicament.

Definitions

[0083] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0084] The term “a” and “an” refers to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. [0085] As used herein, “about” can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art.

[0086] As used herein the specification, “subject” or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals. “Patients” are subjects suffering from or at risk of developing a disease, disorder, or condition or otherwise in need of the compositions and methods provided herein.

[0087] As used herein, “treating” or “treatment” refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. As used herein, “treat or prevent” is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.

[0088] As used herein, “preventing” refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.

[0089] The term “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope. One example of an antigen-binding domain is an antigenbinding domain formed by a VH -VL dimer of an antibody. Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin™.

[0090] As used herein, a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non- beneficial event to the individual to whom the composition is administered. By “therapeutically effective dose” herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0091] As used herein, a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell. The term “TFP” is used interchangeably herein with “TRuC” (T cell receptor fusion construct). A “TFP T cell” or “TRuC T cell” is a T cell that has been transduced (e.g., according to the methods disclosed herein) and that expresses a TFP/TRuC, e.g., incorporated into the natural TCR; thus, a TFP T cell or TRuC T cell is a T cell that has been transduced with a TFP (TRuC). In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell. In some embodiments, the TFP T cell is an NK cell. . In some embodiments, the TFP T cell is an alpha-beta T cell. In some embodiments, the TFP T cell is a gamma-delta T cell.

[0092] The term “PD-1 Switch” or “PD-1 switch receptor” or “PD-1 fusion protein” and the like, as used herein, refers to the described PD-1 fusion proteins that receive an inhibitory signal by binding to PD-L1 or PD-L2, and transform (i.e., "switch") the signal via the co-stimulatory polypeptide of the fusion protein into an activating signal. Thus, in some embodiments, the PD-1 Switch comprises an extracellular PD-1 polypeptide, a transmembrane domain polypeptide, and an intracellular costimulatory polypeptide. In some embodiments, the intracellular costimulatory polypeptide is the intracellular signaling domain of a co-stimulatory domain, such as, for example, the intracellular signaling domain of 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, or FcyRIII. In certain embodiments, the PD-1 switch is a “PD-lxCD28 switch receptor,” which as used herein refers to a polypeptide comprising a PD-1 extracellular domain, a PD- 1 transmembrane domain, and a CD28 intracellular domain.

[0093] The term "co-stimulatory domain" as used herein refers to the cognate binding partner on a T- cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T- cell, such as, but not limited to, proliferation. Co-stimulatory molecules comprise cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response and include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP10, DAP 12, CD30, LIGHT, 0X40, GITR, CD2, CD27, CD7, CD28, CDS, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1, also known as CD1 la/CD18), NKG2C, ICOS, BAFFR, HVEM, NKG2C, SLAMF7, NKp80, CD160, B7-H3, 4-1BB (CD137), and a ligand that specifically binds with CD83. Costimulatory molecules also comprise an intracellular signaling domain. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.

[0094] The term “2A” “2A self-cleaving peptide,” or “2A peptide,” as used herein, refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP. “P2A” (or “P2AW”) refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP. “E2A” refers to the 2 A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP. F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP. In some embodiments, adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency (e.g., GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 59).

[0095] As used herein, the term “mesothelin” also known as MSLN or CAK1 antigen or Pre-pro- megakaryocyte-potentiating factor, refers to the protein that in humans is encoded by the MSLN (or Megakaryocyte-potentiating factor (MPF)) gene. Mesothelin is a 40 kDa protein present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. The mesothelin gene encodes a precursor protein that is processed to yield mesothelin which is attached to the cell membrane by a glycophosphatidylinositol linkage and a 31-kDa shed fragment named megakaryocyte-potentiating factor (MPF). Mesothelin may be involved in cell adhesion, but its biological function is not known. Mesothelin is a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. Mesothelin is an antigenic determinant detectable on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and some squamous cell carcinomas (see, e.g., Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. Cancer Res. 12:4225-4231(2006)). Mesothelin interacts with CA125/MUC16 (see, e.g., Rump et al., J. Biol. Chem. 279:9190-9198(2004) and Ma et al., J. Biol. Chem. 287:33123-33131(2012)).

[0096] The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human mesothelin can be found as UniProt/Swiss-Prot Accession No. Q13421. The human mesothelin polypeptide canonical sequence is UniProt Accession No. Q13421 (or Q13421-1): MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS LSP RQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLL LF LNPD AF SGPQ ACTRFF SRITKANVDLLPRGAPERQRLLP A ALAC WG VRGSLLSE AD VR ALG GLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMD A LRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKK AREI DESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQH L GYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQ L DKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQ NM NGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHV E GLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGP V LTVLALLLASTLA.

[0097] The nucleotide sequence encoding human mesothelin transcript variant 1 can be found at Accession No. NM005823. The nucleotide sequence encoding human mesothelin transcript variant 2 can be found at Accession No. NM013404. The nucleotide sequence encoding human mesothelin transcript variant 3 can be found at Accession No. NM001177355. Mesothelin is expressed on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and squamous cell carcinomas (see, e.g., Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. Cancer Res. 12:4225-4231(2006)). Other cells that express mesothelin are provided below in the definition of “disease associated with expression of mesothelin.” Mesothelin also interacts with CA125/MUC16 (see, e.g., Rump et al., J. Biol. Chem. 279:9190-9198(2004) and Ma et al., J. Biol. Chem. 287:33123-33131(2012)). In one example, the antigen-binding portion of TFPs recognizes and binds an epitope within the extracellular domain of the mesothelin protein as expressed on a normal or malignant mesothelioma cell, ovarian cancer cell, pancreatic adenocarcinoma cell, or squamous cell carcinoma cell.

[0098] The term “antibody,” as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.

[0099] The terms “antibody fragment” or “antibody binding domain” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[00100] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[00101] “Heavy chain variable region” or “VH” ” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs. [00102] Unless specified, as used herein a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[00103] The portion of the TFP composition of the disclosure comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a TFP composition of the disclosure comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.

[00104] The term “antigen” or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.

[00105] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[00106] The term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the disclosure in prevention of the occurrence of tumor in the first place.

[00107] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[00108] The term “allogeneic” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[00109] The term “xenogeneic” refers to a graft derived from an animal of a different species. [00110] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer, and the like.

[00111] The phrase “disease associated with expression of mesothelin” includes, but is not limited to, a disease associated with expression of mesothelin, or condition associated with cells which express mesothelin including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is a pancreatic cancer. In one aspect, the cancer is an ovarian cancer. In one aspect, the cancer is a stomach cancer. In one aspect, the cancer is a lung cancer. In one aspect, the cancer is an endometrial cancer. Non-cancer related indications associated with expression of mesothelin include, but are not limited to, e.g., autoimmune disease, (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergy and asthma), and transplantation.

[00112] The term “unresectable” as used herein refers to a tumor lesion in which clear surgical excision margins cannot be obtained without leading to significant functional compromise.

[00113] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a TFP of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.

[00114] The term “stimulation” refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[00115] The term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “IT AM”. Examples of an IT AM containing primary cytoplasmic signaling sequence that is of particular use in the disclosure includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.

[00116] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[00117] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell. Examples of immune effector function, e.g., in a TFP-expressing T cell, include cytolytic activity and T helper cell activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.

[00118] A primary intracellular signaling domain can comprise an IT AM (“immunoreceptor tyrosinebased activation motif’). Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[00119] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[00120] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain one or more introns.

[00121] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.

[00122] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[00123] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[00124] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[00125] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[00126] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[00127] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[00128] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[00129] The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[00130] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary- determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antib ody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[00131] “Human” or “fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[00132] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[00133] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[00134] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[00135] The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.

[00136] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[00137] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[00138] The term “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[00139] The term “promoter/regulatory sequence” refers to a nucleic acid sequence which may be required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[00140] The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[00141] The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. [00142] The term “tissue-specific” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[00143] The terms “linker” and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly- Ser) _n , where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (GlysSer). Also included within the scope of the disclosure are linkers described in WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises (G4S) _n , wherein n=l to 3.

[00144] As used herein, a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription. The 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co- transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

[00145] As used herein, “/// vitro transcribed RNA” refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

[00146] As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation. [00147] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3’ end. The 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[00148] As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[00149] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[00150] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human). Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. A “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.

[00151] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro. [00152] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[00153] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[00154] In the context of the present disclosure, “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, gastric cancer, ovarian cancer, NHL, leukemia, uterine cancer, prostate cancer, colon cancer, cervical cancer, bladder cancer, kidney cancer, brain cancer, liver cancer, pancreatic cancer, brain cancer, endometrial cancer, and stomach cancer.

[00155] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[00156] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[00157] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or greater in a recited variable.

[00158] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.

[00159] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.

[00160] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.

[00161] The term “effector T cell” includes T helper (e.g., CD4+) cells and cytotoxic (e.g., CD8+) T cells. CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.

[00162] The term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells. In some aspects, the regulatory T cell has a CD4+CD25+Foxp3+ phenotype. In some aspects, the regulatory T cell has a CD8+CD25+ phenotype. See Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, incorporated by reference in its entirety. [00163] In some instances, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, malignant pleural disease, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with mesothelin expression, and combinations thereof, a disease associated with mesothelin expression, and combinations thereof.

[00164] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[00165] The term “specifically binds,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g. , mesothelin) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.

[00166] The term “line of therapy,” as used herein, refers to a treatment that consists of one or more complete treatment cycles with a single agent, surgery, or ration therapy, a regimen consisting of a combination of several drugs, surgery, or radiation therapy, or a planned sequential therapy of various regimens. A treatment is considered a new line of therapy if any one of the following two conditions are met:

(i) Start of a new line of treatment after discontinuation of a previous line of treatment: If a treatment regimen is discontinued for any reason and a different regimen is started, it should be considered a new line of therapy. A regimen is considered to have been discontinued if all the drugs, radiation therapy or surgery in that given regimen have been stopped. A regimen is not considered to have been discontinued if some of the drugs, radiation therapy, or surgery of the regimen, but not all, have been discontinued.

(ii) The unplanned addition or substitution of one or more drugs, radiation therapy, or surgery in an existing regimen: Unplanned addition of a new drug, a new radiation therapy, or a new surgery or unplanned switching to a different drug (or combination of drugs), a different radiation therapy, or a different surgery for any reason is considered a new line of therapy.

[00167] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

Mesothelin

[00168] Mesothelin is a 40 kDa glycosyl-phosphatidyl inositol-linked membrane protein differentiation antigen, whose expression is mostly restricted to mesothelial cells lining the pleura, pericardium and peritoneum in healthy individuals (Chang and Pastan, 1996; Chang et al, 1992; Hassan and Ho, 2008). Mesothelin is overexpressed in multiple cancers, including more than 90% of malignant pleural mesotheliomas (MPMs) and pancreatic adenocarcinomas, approximately 70% of ovarian cancers, and approximately half of non-small cell lung cancers (NSCLCs), among others (Argani et al, 2001; Hassan and Ho, 2008; Hassan et al, 2005; Ordonez, 2003). The precise physiological function of mesothelin is not completely understood, but it has been postulated to promote metastasis through its binding to MUC16 (Chen et al, 2013). MSLN (the gene encoding for mesothelin) knockout mice grow and reproduce normally and have no detectable phenotype. Therapeutic modalities include antibodies, recombinant immunotoxins, and CAR T cells. However, aberrant mesothelin expression plays an active role in both malignant transformation and tumor aggressiveness by promoting cancer cell proliferation, invasion, and metastasis.

[00169] Mesothelin expression is normally restricted to serosal cells of the pleura, peritoneum, and pericardium. Mesothelin is highly expressed in a wide range of solid tumors, including epithelioid mesothelioma (95%), extrahepatic biliary cancer (95%), pancreatic adenocarcinoma (85%), serous ovarian adenocarcinoma (75%), lung adenocarcinoma (57%), triple negative breast cancer (66%), endometrial carcinoma (59%), gastric carcinoma (47%), colorectal carcinoma (30%), and others. [00170] Mesothelin overexpression is associated with poorer prognosis in mesothelioma, ovarian cancer, cholangiocarcinoma, lung adenocarcinoma, triple-negative breast cancer, and pancreatic adenocarcinoma.

[00171] Given its high expression in tumors and low expression in normal tissue, mesothelin is an attractive target for immunotherapy. Currently, several chimeric antigen receptor (CAR) T cell programs directed against mesothelin are being investigated.

[00172] The compositions and methods comprising the engineered T cells disclosed herein are a novel cell therapy that consists of genetically engineered T cells that express a PD-1 Switch receptor, and an antibody domain (e.g., a single-domain antibody or a single chain Fv) that recognizes human mesothelin fused to a TCR subunit (e.g., TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 6, CD3 a, or CD3 C, subunit) which, upon expression, can be incorporated into the endogenous T cell receptor complex. The antibody domain can comprise an anti-MSLN antigen binding domain. The antibody domain can be a murine, human or humanized antibody domain. The anti-MSLN binding domain can be a scFv or a VHH domain. The anti-MSLN binding domain can comprise a domain having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence of an anti-MSLN binding domain disclosed herein, e.g., in Table 6. The anti-MSLN binding domain can comprise a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. The anti-MSLN binding domain can comprise a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, or SEQ ID NO: 31. In some embodiments, the anti-MSLN binding domain comprises a single-domain antibody comprising a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the anti-MSLN binding domain comprises a CDR1 having an amino acid sequence of SEQ ID NO: 37, a CDR2 having an amino acid sequence of SEQ ID NO: 38, and a CDR3 having an amino acid sequence of SEQ ID NO: 39.

Mesothelin Expression in Cancer

[00173] The expression of mesothelin in cancer has been broadly studied. Serial analyses of gene expression (SAGE: www.ncbi.nlm.nih.gov/projects/SAGE/), conducted at the National Institutes of Health (NIH), have shown high messenger ribonucleic acid (mRNA) expression of mesothelin in NSCLC, pancreatic cancer, MPM, ovarian cancer, cholangiocarcinoma, and other adenocarcinomas (Hassan and Ho, 2008). Expression profiles have been further supported by immunohistochemistry (IHC) studies performed on biopsy tissues taken from patients with multiple tumor types (Inaguma et al, 2017). While the IHC staining can vary dependent on antibody clone that is used, most IHC analyses indicate that 90% of ovarian cancer and > 75% of MPM or pancreatic cancer biopsies are immunoreactive to anti-mesothelin antibodies. Mesothelin expression and prevalence in various tumor types has been reviewed by Morello et al (2016) (Table 1).

Malignant Pleural Mesothelioma (MPM)

[00174] MPM represents about 80% of mesothelioma cases. MPM is a regional and highly aggressive tumor that arises from the mesothelium of the pleural surface. Rarely, other serosal membranes of the human body are also coated with mesothelium, such as peritoneum (peritoneal mesothelioma) and pericardium (pericardial mesothelioma), are affected. The incidence of MPM has increased significantly and it is estimated that 40,000 people die each year worldwide due to asbestos-related MPM. Different types of MPM have been identified including epithelioid (50%-70% of cases), biphasic (30%), and sarcomatoid (10%-20%) with increasingly aggressive behavior and worse prognosis. In addition to a high incidence (25%-60%) of somatic BAP1 mutations, MPM is also associated with frequent alterations in other major tumor suppressor genes, such as pl6/Cdkn2a, pl9/Arf, pl9/Cdkn2b, and NF2. Effective treatment options for patients with MPM are very limited. The standard of care recommended for MPM is palliative chemotherapy with a doublet of platinum salt and an anti-folate. Unfortunately, objective response rates are 17% to 40% and the median overall survival (OS) of patients with MPM is 12 to 19 months when systemic chemotherapy is used with or without anti-angiogenic agents or targeted therapy. Anti-CTLA-4 failed to show a survival advantage as second-line therapy in MPM. Anti -programmed death receptor- 1 (PD-1) and anti-PD-Ll antibodies (e.g., pembrolizumab, nivolumab, avelumab) are currently being tested in several trials in MPM. Early phase trials with anti-PD-1 or anti-PD-Ll antibodies have shown partial response rates up to 28% and disease control rates up to 76% with median duration of response of 12 months, but confirmatory data are required to validate these agents as the second line treatment of choice in MPM.

Pancreatic Cancer

[00175] Pancreatic ductal adenocarcinoma is the seventh leading cause of cancer death worldwide and has the highest incidence-to-mortality ratio of any solid tumor. The median 5-year survival among patients with pancreatic cancer is only 9%, highlighting the urgent need to develop novel therapeutic strategies for this deadly disease. The mainstay of current treatment for metastatic pancreatic ductal adenocarcinoma is combination chemotherapy. There are 2 regimens that are first-line treatment options for patients with metastatic disease with good performance status: FOLFIRINOX (fluorouracil, leucovorin, irinotecan, and oxaliplatin) and gemcitabine combined with nanoparticle albumin-bound paclitaxel (nab-paclitaxel). The EGFR inhibitor erlotinib was tested in combination with gemcitabine for the first-line treatment of unselected patients with metastatic pancreatic ductal adenocarcinoma and showed a statistically but not clinically significant increased OS (6.2 months vs 5.9 months), leading to its FDA approval. However, as other regimens were shown to have more promising results, this combination is rarely utilized. The only approved second-line therapy is a combination of fluorouracil and protein-encapsulated irinotecan for patients who received a gemcitabine-based therapy in the first line. In practice, however, patients with good performance status are often offered the alternative first-line regimen that they did not receive initially. Other actionable mutations found to be enriched in small patient subsets with KRAS wild-type pancreatic cancer, including anaplastic lymphoma kinase (ALK) amplifications (0.16%), BRAF mutations (2.2%), NRG1 fusions (0.5%), and NTRK gene fusions (0.3%).

Triple Negative Breast Cancer (TNBC)

[00176] TNBC fails to express the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor-2 (HER-2) and represents approximately 15% of all breast cancers. Historically, the treatment of TNBC has been limited to chemotherapy given the lack of standard therapeutic targets. Early stage TNBC has a high response rate to chemotherapy, but relapse is common and tends to occur rapidly. Once TNBC becomes metastatic, it is incurable. Single agent chemotherapy with an anthracy cline, a taxane, or a platinum agent are the most active regiments in the frontline setting in metastatic TNBC, with response rates in 20%-30% of cases and a median OS of 10-13 months. Combination chemotherapy regimens can increase ORRs, but they are also associated with more toxicity without clear evidence of improvement on OS. Transcriptomic analyses have more precisely classified breast cancers into distinct molecular subtypes, including normal breast-like, luminal A and luminal B (ER + and/or PgR+), HER-2 enriched, claudin-low, and basal-like. Most cases of TNBC (50%-75%) are basal-like, whereas those with low levels of ER and PgR expression (1%-10%) are more likely to be luminal (46%) or HER-2-enriched (29%) by gene expression. Most claudin-low breast tumors fail to express ER, PgR, and HER-2 by IHC and have metaplastic/medullary differentiation, elevated expression of immune-related genes, stem cell and mesenchymal features, and active transforming growth factor-beta signaling.

[00177] Further analyses have defined additional subtypes, including 2 basal-like (BL1 and BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem- like (MSL), luminal androgen receptor (LAR) and unstable (UNS) subtypes, based on tumor cell intrinsic gene expression patterns. Recent progress has identified 2 groups of patients with metastatic TNBC for whom biomarker-driven targeted therapy has been approved by the FDA. First, patients with germline BRCA1/2 mutations (20% of TNBC cases), for whom the PARP inhibitors Olaparib (ORR 59.9%, PFS 7 months, OS 17.1 months in patients with 2 or fewer prior lines of therapy) and talazoparib (ORR 62.6%, PFS 8.6 months in patients with 3 or fewer prior lines of therapy) are approved after progression on chemotherapy, and second, patients with tumors that express PD-L1 on immune cells occupying at least 1% of the tumor area, for whom immunotherapy with the PD-L1 inhibitor atezolizumab in combination with nab- paclitaxel (ORR 59%, PFS 7.5 months, OS 25 months in treatment naive patients) is approved.

Colorectal Cancer

[00178] Colorectal cancer (CRC) is the second greatest cancer threat to life in Western countries. Metastatic colorectal cancer carries poor prognosis, and current therapeutic regimes convey limited improvements in survival and high rates of detrimental side effects in patients that may not stand to benefit. Little progress has been made regarding targeted therapies for patients with metastatic colorectal cancer (mCRC). The CALGB/SWOG 80405, Fire-3, and PEAK randomized controlled clinical trials have established the vascular endothelial growth factor receptor (VEGFR) inhibitor bevacizumab and the EGFR inhibitors cetuximab and panitumumab as frontline therapies for patients with mCRC when combined with FOLFOX/FOLFOXIRI chemotherapy. However, the improvement in outcomes standard regimens has been modest, with PFS in FIRE-3 improved from 5.8 months to 9.7 months with the addition of cetuximab in patients with wild-type RAS CRC. Recently, the Phase III Keynote-177 study compared the clinical activity of pembrolizumab to that of standard 5FU-based chemotherapy (with or without bevacizumab or cetuximab) among 307 patients with newly diagnosed measles, mumps, rubella (MMR) deficient/MSI-H CRC. Pembrolizumab was superior to chemotherapy with PFS of 16.6 months compared with 8.2 months. The median OS had not yet been reached in the pembrolizumab group but was 36.7 months in the chemotherapy group. An overall response rate was 43.8% in the pembrolizumab group, compared with 33.1% in the chemotherapy group. These data have established checkpoint inhibitor therapy as standard of care among patients with newly diagnosed MMR deficient/MSI-high CRC. However, the benefit of this approach is very limited among late-stage patients refractory to chemotherapeutic regimens.

Non-small Cell Lung Cancer

[00179] NSCLC remains the leading cause of cancer-related mortality worldwide, accounting for approximately 18% of all cancer deaths. Despite treatment with platinum- and taxane-based chemotherapy, patients with metastatic NSCLC have a median survival of approximately 10 months, and a 5 -year survival rate of approximately 15%. Despite the increased number of treatment options available for patients with non-squamous histology NSCLC, there has been little OS improvement from several new agents, including pemetrexed, erlotinib, and bevacizumab beyond very small subpopulations. Therapeutic options for mutation wild-type non-squamous NSCLC are particularly limited after failure of front-line chemotherapy. Overall, this group of patients only has an OS of about 8 months after progression from platinum agents. Once resistance to tyrosine kinase inhibitors (TKIs) occurs, the patients who have epidermal growth factor receptor (EGFR) mutations or ALK translocations will have a rapid disease progression. Therefore, NSCLC remains a disease with high unmet medical need. Recently, T cell checkpoint regulators such as CTLA-4 and programmed death- 1 (PD-1, CD279) down- regulate T cell activation and proliferation upon engagement by their cognate ligands. T cell checkpoint inhibitors induce antitumor activity by breaking immune tolerance to tumor cell antigens. PD-1 and PD-L1 inhibitors are effective against metastatic NSCLC lacking sensitizing EGFR or ALK mutations.

[00180] Pembrolizumab (Keytruda®, Merck), nivolumab (Opdivo®, Bristol-Myers Squibb), and atezolizumab (Tecentriq®, Genentech) are approved as second-line therapy. Among patients in whom the percentage of tumor cells with membranous PD-L1 staining (tumor proportion score) is 50% or greater, pembrolizumab has also replaced cytotoxic chemotherapy as the first-line treatment of choice. However, patients with a tumor proportion score of 50% or greater represent a minority of those with NSCLC. A randomized, phase 2 trial of carboplatin plus pemetrexed with or without pembrolizumab showed significantly better rates of response and longer progression-free survival (PFS) with the addition of pembrolizumab than with chemotherapy alone. In the global, double-blind, placebo- controlled, phase 3 KEYNOTE- 189 trial, the addition of pembrolizumab to standard chemotherapy of pemetrexed and a platinum-based drug resulted in significantly longer OS and PFS than chemotherapy alone and such combination is likely to become standard frontline therapy (Ghandi et al, 2018). Notably, no standard of care is available for patients failing to respond or relapsing after checkpoint inhibitor therapy.

Ovarian Cancer

[00181] Ovarian cancers can be classified in several subtypes according to their histopathology, which also determines their therapy. Epithelial ovarian cancer comprises 90% of all ovarian malignancies, with other pathologic subtypes such as germ cell and sex-cord stromal tumors being much rarer. It is estimated that 22,240 new diagnoses and 14,070 deaths from ovarian cancer will occur in 2018 in the United States (SEER, 2018). Ovarian cancer is characterized by late-stage presentation (more than 70% of cases), bulky metastatic tumor burden, and frequent recurrence of eventual chemoresistant disease, which result in cure rates below 15% among subjects with stage 3/4 disease. The 2 canonical types of drugs used to treat ovarian cancer — taxane and platinum-based agents — have not been replaced in the past 20 years, although the optimum timing of treatment (neoadjuvant versus adjuvant) and the best route of administration (intravenous versus intraperitoneal) remain unknown.

[00182] Recurrent ovarian cancer is not curable. The objectives of therapy are symptom palliation and extension of life. Subjects with platinum-sensitive ovarian cancer should be treated with a platinumbased agent. Those progressing after platinum retreatment and those with platinum-resistant disease, non-platinum combination and targeted therapies are available. The initial clinical efficacy of novel therapeutics, such as poly(ADP-ribose) polymerase (PARP) inhibitors and immune-checkpoint inhibitors, has ushered in a new wave of drug development in ovarian cancer. The synthetic lethality of BRCA mutated (e.g., deficient) ovarian cancer cells exposed to the PARP inhibitor olaparib resulted in a median PFS of 7 months and median OS of 16.6 months. Efficacy with checkpoint inhibitors in subjects with advanced recurrent ovarian cancer has been modest thus far. Best overall response rate (ORR) has been 15% with nivolumab, 12% with pembrolizumab, and 10% with ipilimumab (Hamanishi et al, 2015; Varga et al, 2015).

Cholangiocarcinoma

[00183] Cholangiocarcinomas are biliary epithelial tumors of the intrahepatic, perihilar, and distal biliary tree. Intrahepatic cholangiocarcinomas (iCCAs) (20% of cases) arise above the second-order bile ducts, whereas the cystic duct is the anatomical point of distinction between perihilar cholangiocarcinomas (pCCAs) (50%-60%), and distal cholangiocarcinomas (dCCAs; 20-30%). Most subjects have advanced- stage disease at presentation due to its aggressiveness and difficulty in early diagnosis. While surgery is the preferred therapy, only 35% of cases have early disease amenable to surgical resection with curative intent. For unresectable cholangiocarcinoma, the available standard- of-care chemotherapy (gemcitabine and cisplatin) renders a median OS < 1 year, partly due to the desmoplastic stroma that fosters cancer cell survival and poses a barrier to the delivery of chemotherapy. Recurrent mutations in IDH1, IDH2, FGFR1, FGFR2, FGFR3, EPHA2, and BAP1 are found predominantly in iCCAs, whereas ARID1B, ELF3, PBRM1, PRKACA, and PRKACB mutations occur preferentially in pCCA/dCCA. Some of the latter represent actionable mutations whose therapeutic potential is currently being investigated in clinical trials. At present, clinical data on immunotherapy in cholangiocarcinoma are limited. PD-L1 expression has been reported in 9% to 72% of specimens, and on 46% to 63% of immune cells within the tumor microenvironment. Interim data from the KEYNOTE-028 basket trial (NCT02054806) with pembrolizumab have been reported. Of the 24 enrolled subjects with PD-L1 expression >1% (20 cholangiocarcinoma, 4 gallbladder carcinoma), 4 (17%, 3 with cholangiocarcinoma and 1 with gallbladder carcinoma) had a partial response (PR), and 4 (17%) had stable disease (SD). The median PFS was not reached at the time of reporting. These data prompted the launching of a biliary cancer cohort of 100 subjects in the ongoing KEYNOTE-158 basket trial (NCT02628067).

PD-1 fusion protein

[00184] The present disclosure encompasses DNA and RNA constructs encoding PD-1 fusion proteins, wherein the fusion protein comprises an extracellular domain of PD-1 or a portion thereof capable of interacting with a ligand of PD-1 (e.g., PDL1 and/or PDL2), and an intracellular signaling domain. In some embodiments, the PD-1 fusion protein is referred to herein as a “PD-1 switch receptor”. In embodiments, the intracellular signaling domain is a costimulatory domain. In some embodiments the costimulatory domain is selected from the group consisting of 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the PD-1 fusion protein comprises a CD28 costimulatory domain. Such PD-1 fusion proteins are referred to herein as a “PD-lxCD28 switch receptor,” which term may be used interchangeably herein with the term “PD-1-CD28 fusion protein” or “PD-1-CD28 switch receptor” or "PD-lxCD28 fusion protein”.

[00185] In some embodiments, the PD-1 switch receptor comprises a PD-1 extracellular domain (ED; also referred to herein as PD-1 EC) or a portion thereof. In some embodiments, the PD-1 switch receptor comprises a PD-1 ED comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 99% sequence identity to SEQ ID NO: 54. In some embodiments, the PD-1 switch receptor comprises a PD-1 ED comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the PD-1 switch receptor comprises a PD-1 transmembrane domain ™ comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 99% sequence identity to SEQ ID NO: 55. In some embodiments, the PD-1 switch receptor comprises a PD-1 TM comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the PD-1 switch receptor comprises a PD-1 ED and a PD-1 transmembrane domain (TM). In some embodiments, the PD-1 switch receptor comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 99% sequence identity to SEQ ID NO: 56. In some embodiments, the PD-1 switch receptor comprises the amino acid sequence of SEQ ID NO: 56.

[00186] The PD-1 switch receptor can be a PD-lxCD28 Switch receptor. In some embodiments, the PD-1 switch receptor comprises a CD28 intracellular domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 99% sequence identity to SEQ ID NO: 57. In some embodiments, the PD-1 switch receptor comprises a CD28 intracellular domain comprising the amino acid sequence of SEQ ID NO: 57. In some embodiments, the PD-lxCD28 switch receptor comprises a PD-1 extracellular domain, a PD-1 transmembrane domain, and a CD28 intracellular signaling domain. In some embodiments, the PD-lxCD28 switch receptor is a fusion protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 58. In some embodiments, the PD- lxCD28 switch receptor is a fusion protein comprising the amino acid of SEQ ID NO: 58.

T cell receptor (TCR) fusion proteins (TFPs)

[00187] The present disclosure encompasses DNA and RNA constructs encoding TFPs (e.g., anti- MSLN TFPs), and variants thereof, wherein the TFP comprises a binding domain, e.g., an antibody or an antibody fragment, a ligand, or a ligand binding protein, that binds specifically to a tumor-associated antigen e.g., mesothelin, e.g., human mesothelin, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. The TFPs are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.

[00188] The TFPs can comprise a target-specific binding element otherwise referred to as an antigen binding domain. The choice of moiety depends upon the type and number of target antigen that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the disclosure include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases). [00189] The TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen. A portion of the TFP may comprise the antigen binding domain that targets mesothelin.

[00190] The antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like. Likewise, a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP. In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the TFP will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the TFP to comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.

[00191] Thus, the antigen-binding domain can comprise a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. The humanized or human anti- mesothelin binding domain may comprise one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-mesothelin binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti- mesothelin binding domain described herein, e.g., a humanized or human anti-mesothelin binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. The humanized or human anti-mesothelin binding domain may comprise one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., the humanized or human anti-mesothelin binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. The humanized or human anti-mesothelin binding domain may comprise a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. The humanized or human anti-mesothelin binding domain may comprise a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. The anti- mesothelin binding domain can be a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein. The anti-mesothelin binding domain can be a VHH comprising a heavy chain of an amino acid sequence provided herein. The anti-mesothelin binding domain (e.g., a scFv or VHH) may comprise: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications e.g., substitutions) but not more than 30, 20 or 10 modifications e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications e.g., substitutions) but not more than 30, 20 or 10 modifications e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. The humanized or human anti-mesothelin binding domain can be a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein. The humanized anti-mesothelin binding domain may include a (Gly4-Ser) _n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S) _n , wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S) _n , wherein n=l to 3.

[00192] A non-human antibody may be humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.

[00193] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91 :969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s- 5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties).

[00194] A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanized antibodies or antibody fragments may comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, e.g., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety. [00195] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151 :2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). The framework region, e.g., all four framework regions, of the heavy chain variable region may be derived from a VH4-4-59 germline sequence. The framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence. The framework region, e.g., all four framework regions of the light chain variable region may be derived from a VK3-1.25 germline sequence. The framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.

[00196] The portion of a TFP composition that comprises an antibody fragment can be humanized with retention of high affinity for the target antigen and other favorable biological properties. Humanized antibodies and antibody fragments may be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[00197] A humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind human mesothelin. A humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to human mesothelin.

[00198] The anti-mesothelin binding domain can be characterized by particular functional features or properties of an antibody or antibody fragment. For example, the portion of a TFP composition of the disclosure that comprises an antigen binding domain can specifically bind human mesothelin. The antigen binding domain has the same or a similar binding specificity to human mesothelin as the FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). The disclosure can relate to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a mesothelin protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence provided herein. The scFv may be contiguous with and in the same reading frame as a leader sequence.

Stability and Mutations

[00199] The stability of an anti-mesothelin binding domain, e.g., sdAb or scFv molecules (e.g., soluble sdAb or scFv) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full-length antibody. The humanized or human sdAb or scFv may have a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent sdAb or scFv in the described assays.

[00200] The improved thermal stability of the anti-mesothelin binding domain, e.g., sdAb or scFv is subsequently conferred to the entire mesothelin-TFP construct, leading to improved therapeutic properties of the anti-mesothelin TFP construct. The thermal stability of the anti-mesothelin binding domain, e.g., sdAb or scFv can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody. The anti-mesothelin binding domain, e.g., sdAb or scFv may have a 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, or 15 °C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the sdAb or scFv molecules disclosed herein and sdAb or scFv molecules or Fab fragments of an antibody from which the sdAb VHH was derived or the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described below. [00201] Mutations in sdAb or scFv (arising through humanization or mutagenesis of the soluble sdAb or scFv) alter the stability of the sdAb or scFv and improve the overall stability of the sdAb or scFv and the anti-mesothelin TFP construct. Stability of the humanized scFv is compared against the llama sdAb or murine scFv using measurements such as TM, temperature denaturation and temperature aggregation. The anti-mesothelin binding domain, e.g., a sdAb or scFv, may comprise at least one mutation arising from the humanization process such that the mutated sdAb or scFv confers improved stability to the anti-mesothelin TFP construct. The anti-mesothelin binding domain, e.g., sdAb or scFv may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated sdAb or scFv confers improved stability to the mesothelin-TFP construct.

[00202] The antigen binding domain of the TFP may comprise an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-mesothelin antibody fragments described herein. The TFP composition of the disclosure may comprise an antibody fragment, e.g., a sdAb or scFv.

[00203] The antigen binding domain of the TFP can be engineered by modifying one or more amino acids within one or both variable regions (e.g., VHH, VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. The TFP composition of the disclosure may comprise an antibody fragment, e.g., a sdAb or scFv.

[00204] It will be understood by one of ordinary skill in the art that the antibody or antibody fragment of the TFP may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity. For example, additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein. For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. A string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.

[00205] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). [00206] Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[00207] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[00208] The present disclosure contemplates modifications of the starting antibody or fragment (e.g., sdAb or scFv) amino acid sequence that generate functionally equivalent molecules. For example, the VHH and VH or VL of an anti-mesothelin binding domain, e.g., sdAb or scFv, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VHH and VH or VL framework region of the anti-mesothelin binding domain, e.g., sdAb or scFv. The present disclosure contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules. The TFP construct can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the starting TFP construct.

Extracellular domain of the TFP

[00209] The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain. An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g., the alpha, beta, gamma, or delta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some instances, the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[00210] In some embodiments, the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the TCR extracellular domain comprises an IgC domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.

[00211] In some embodiments, the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[00212] In some embodiments, the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of an IgC domain of

TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[00213] In some embodiments, the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,

91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C- terminus.

Transmembrane Domain of the TFP

[00214] In general, a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region). In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface. In a different aspect the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.

[00215] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target. In some instances, the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[00216] In some embodiments, the transmembrane domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[00217] In some instances, the transmembrane domain can be attached to the extracellular region of the TFP, e.g., the antigen binding domain of the TFP, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

Linkers

[00218] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the binding element and the TCR extracellular domain of the TFP. A glycineserine doublet provides a particularly suitable linker. In some cases, the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, X is 2. In some embodiments, X is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC .

Cytoplasmic Domain of the TFP

[00219] The cytoplasmic domain of the TFP can include an intracellular domain. In some embodiments, the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta. In some embodiments, the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00220] Examples of intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.

[00221] In some embodiments, the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.

[00222] In some embodiments, the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C- terminus or at both the N- and C-terminus.

[00223] In some embodiments, the intracellular domain comprises, or comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 or more consecutive amino acid residues of the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.

[00224] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal may be required. Thus, naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).

[00225] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs).

[00226] Examples of IT AMs containing primary intracellular signaling domains that are of particular use in the present disclosure include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 epsilon, CD3 delta, or CD3 gamma. In one embodiment, a primary signaling domain comprises a modified IT AM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[00227] The intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure. For example, the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that may be required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012; 119(3):696-706).

[00228] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.

[00229] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[00230] In one aspect, the TFP-expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., MSLN) or a different target (e.g., CD70, CD19, or MUC16). In one embodiment, when the TFP- expressing cell comprises two or more different TFPs, the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another. For example, a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not form an association with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.

[00231] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD-1, can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, LAG3, CTLA4, CD 160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD-1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). PD- 1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Int. Immunol 8:765-75). Two ligands for PD-1, PD-L1 and PD-L2, have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al., 2000 J. Exp. Med. 192: 1027-34; Latchman et al., 2001 Nat. Immunol. 2:261-8; Carter et al., 2002 Eur. J. Immunol. 32:634-43). PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med. 81 :281-7; Blank et al., 2005 Cancer Immunol. Immunother. 54:307-314; Konishi et al., 2004 Clin. Cancer Res. 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1.

[00232] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD-1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD-1 TFP). In one embodiment, the PD-1 TFP, when used in combinations with an anti-MSLN TFP described herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD-1 TFP comprising the extracellular domain of PD-1. Alternatively, provided are TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death-Ligand 2 (PD-L2).

[00233] In another aspect, the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP-T cells can include a first cell expressing a TFP having an anti-MSLN binding domain described herein, and a second cell expressing a TFP having a binding domain specifically targeting a different antigen, e.g., a binding domain described herein that differs from the anti-MSLN binding domain in the TFP expressed by the first cell. As another example, the population of TFP-expressing cells can include a first cell expressing a TFP that includes a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen).

[00234] In another aspect, the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In some embodiments, the agent is a cytokine. In some embodiments, the cytokine is IL-15. In some embodiments, IL-15 increases the persistence of the T cells described herein.

Recombinant Nucleic Acids Encoding a TFP and/or PD-1 fusion protein

[00235] Disclosed herein, in some embodiments, are recombinant nucleic acids encoding the TFPs and PD-1 fusion proteins disclosed herein. In an aspect, provided herein is a composition comprising a first isolated recombinant nucleic acid molecule encoding a first fusion protein comprising a TCR subunit comprising at least a portion of a TCR extracellular domain, and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of a CD3 subunit; and a first target binding domain, wherein the TCR subunit and the first target binding domain are operatively linked, and wherein the first fusion protein incorporates into a TCR when expressed in a T-cell; and a second isolated recombinant nucleic acid molecule encoding a second fusion protein having a second target binding domain, wherein the second target binding domain comprises a PD-1 polypeptide which is operably linked to an intracellular domain of a costimulatory polypeptide. In some embodiments, the PD-1 polypeptide is operatively linked via its C-terminus to the N-terminus of the intracellular domain of the costimulatory polypeptide. In some embodiments, the PD-1 polypeptide comprises the extracellular domain and the transmembrane domain of PD-1. In some embodiments, the costimulatory polypeptide is CD28. In some embodiments, the first target binding domain is a human or humanized antibody domain that targets MSLN.

[00236] In one aspect, provided herein is a viral vector comprising a first and a second nucleic molecule described herein. In some embodiments, the first isolated recombinant nucleic acid molecule and the second isolated recombinant nucleic acid molecule are contained in a single operon. In some embodiments, the first isolated recombinant nucleic acid molecule and the second isolated recombinant nucleic acid molecule are contained in two separately transcribed operons. In some embodiments, the operon comprises an Ela promoter. In some embodiments, the operons each comprise an Ela promoter. In some embodiments, the viral vector is a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.

[00237] In one aspect, provided herein is a viral vector comprising a first isolated recombinant nucleic acid molecule described herein. In one aspect, provided herein is a viral vector comprising a second isolated recombinant nucleic acid molecule described herein. In one aspect, provided herein is a mixture comprising a viral vector described herein. In one aspect, provided herein is a transduced T cell comprising a composition described herein or a viral vector described herein or a mixture described herein. In one aspect, provided herein is a transduced T cell comprising one or more viral vectors described herein. In some embodiments, the first fusion protein and the second fusion protein are each detectable on the surface of the T cell. In some embodiments, the present disclosure provides methods for administering such transduced T cells to a subject in need thereof.

[00238] The TC-510 (MHl-CD3s-TRuC/PD-lxCD28) lentiviral vector (LVV) transgene sequence comprises a GM-CSFRa signal peptide followed by the anti-mesothelin MH1 single-domain antibody (sdAb) coding sequence, a flexible 20 amino acid glycine/serine linker, the human CD3s coding sequence, a T2A “self-cleaving” peptide, and a PD-lxCD28 switch receptor sequence. The amino acid sequence of the TC-510 transgene and components are provided in Table 6.

[00239] In one aspect, provided herein is an isolated T cell comprising a plurality of polypeptides, a first polypeptide comprising a TCR subunit comprising at least a portion of a TCR extracellular domain, a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon or CD3 gamma; and a first target binding domain (e.g., an anti-MSLN binding domain), wherein the TCR subunit and the first target binding domain are operatively linked, and wherein the first fusion protein is incorporated into a TCR in the T cell; and a second fusion protein having a second target binding domain, wherein the second target binding domain comprises a PD-1 polypeptide which is operably linked to an intracellular domain of a costimulatory polypeptide. In some embodiments, the PD-1 polypeptide is operatively linked via its C-terminus to the N-terminus of the intracellular domain of the costimulatory polypeptide. In some embodiments, the PD-1 polypeptide comprises the extracellular domain and the transmembrane domain of PD-1. In some embodiments, the costimulatory polypeptide is CD28. Accordingly, in some embodiments, the second fusion protein comprises a PD-1 polypeptide which is operably linked via its C-terminus to the N- terminus of the intracellular domain of CD28, wherein the PD-1 polypeptide comprises the extracellular domain and the transmembrane domain of PD-1. In some embodiments, the present disclosure provides methods for administering such isolated T cells to a subject in need thereof.

[00240] In some instances, the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Non-naturally occurring nucleic acids are well known to those of skill in the art. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.

[00241] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs and PD- 1 fusion proteins. The present disclosure also includes a TFP- and/or PD-1 fusion protein-encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by poly A addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the TFP.

[00242] In one aspect, the anti-MSLN TFP and/or PD-1 fusion protein is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the anti-MSLN TFP and/or PD-1 fusion protein is introduced into a T cell for production of a TFP-T cell. In one embodiment, the in vitro transcribed RNA TFP and/or PD-1 fusion protein can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a TFP and/or PD-1 fusion protein of the present disclosure. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[00243] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-compl ementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.

[00244] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

[00245] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. In one embodiment, the 5’ UTR is between one and 3,000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.

[00246] The 5 ’ and 3 ’ UTRs can be the naturally occurring, endogenous 5 ’ and 3 ’ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3 ’UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[00247] In one embodiment, the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5’ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. In other embodiments the 5’ UTR can be 5 ’UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[00248] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[00249] In a preferred embodiment, the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[00250] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).

[00251] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3’ stretch without cloning highly desirable.

[00252] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[00253] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[00254] 5’ caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5’ cap. The 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958- 966 (2005)).

[00255] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[00256] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector™-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[00257] For additional information on making and using TFP T cells, see U.S. Patent Nos. 10,442,849, 10,358,473, 10,358,474, and 10,208,285; and US Patent Publication No. US20210187022; each of which is herein incorporated by reference in its entirety.

Gene Editing of TCR Complex or Endogenous Protein-coding Genes

[00258] In some embodiments, the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., U.S. Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev. Genetics (2010) vl l, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704-716, each incorporated herein by reference. In some embodiments, one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (e.g., are chimeric).

[00259] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as “genome editing.

[00260] In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD-1), and/or other genes.

[00261] Current gene editing technologies comprise meganucleases, zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a doublestranded DNA break (DSB). DSB may then be repaired by either non-homologous end joining (NHEJ) or, when donor DNA is present, homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, nickase nucleases generate singlestranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.

[00262] Genetic modification of genomic DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005) Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair. Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.

[00263] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), NatProtoc. 8:2281-2308; Mali et al. (2013), NatMethods 10:957-63). The CRISPR geneediting technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).

[00264] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9: 1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the type II CRISPR-Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2018) Nat. Commun. 9: 1911). Among these, such as Cast 2a (Cpfl) proteins from Acidaminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.

[00265] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sei. 30(7): 503-522). Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446). In some embodiments, meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.

[00266] In addition to mentioned four major gene editing technologies, chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446). For example, A megaTAL is a single chimeric protein, which is the combination of the easy-to-tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.

[00267] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lenti virus, and retrovirus.

Modified T cells

[00268] Disclosed herein are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein; and the sequence encoding the PD-1 fusion protein of the nucleic acid disclosed herein or a PD-1 fusion protein encoded by the sequence of the nucleic acid disclosed herein. Such T cells may be interchangeably referred to herein as modified T cells, engineered T cells, or transduced T cells. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the sequence encoding the TFP disclosed herein and/or the PD-1 fusion protein disclosed herein or a TFP and/or PD-1 fusion protein encoded by the sequence of the nucleic acid disclosed herein. [00269] In some embodiments, the modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the sequence encoding the TFP disclosed herein and/or the PD-1 fusion protein disclosed herein or a TFP and/or PD-1 fusion protein encoded by the sequence of the nucleic acid disclosed herein.

[00270] In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain. In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain. In some instances, the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell. In some instances, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some instances, the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell. In some instances, the T cell is a human T cell. In some instances, the T cell is a CD8+ or CD4+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the T cell is a TCR alpha-beta T cell. In some instances, the T cell is a TCR gamma-delta T cell. In some instances, one or more of TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No. WO2019173693, which is herein incorporated by reference.

[00271] In some embodiments, the modified T cells are y6 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y6 T cells are V<51+ V<52- y<5 T cells. In some embodiments, the y6 T cells are V51- V<52+ y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52- y<5 T cells.

[00272] In some instances, the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD-1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.

Sources of T cells

[00273] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term “subj ecf ’ is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells can be obtained from a leukopak. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used. In certain aspects of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one aspect of the present disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative aspect, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe® 2991 cell processor, the Baxter Oncology CytoMate, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions.

[00274] After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media. In some embodiments, the transduced cells are resuspended in a cry opreservation media comprising PlasmaLyte A, Cryostor 10, and human serum albumin (HSA). In further embodiments, the transduced cells are resuspended in a cry opreservation media comprising 49% PlasmaLyte A, 50% Cryostor 10, and 1% HSA. In some embodiments, the present disclosure provides a composition comprising transduced cells provided herein (e.g., TC-510), PlasmaLyte A, Cryostor 10, and HSA. [00275] In embodiments, the T cells are aP T cells. In some embodiments, the T cells are y6 T cells. y6 T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.

[00276] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL® gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, CD4+ and CD8+ T cells are isolated with anti-CD4 and anti-CD8 microbeads. In another aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T or Trans-Act® beads, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this present disclosure. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[00277] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

[00278] In one embodiment, a T cell population can be selected that expresses one or more of IFN-y TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO2013126712, which is herein incorporated by reference.

[00279] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 2 billion cells/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, greater than 100 million cells/mL is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00280] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5xlO ⁶ /mL. In other aspects, the concentration used can be from about lxl0 ⁵ /mL to lxlO ⁶ /mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.

[00281] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.

[00282] Also contemplated in the context of the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.

[00283] In a further aspect of the present disclosure, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Activation and Expansion of T Cells

[00284] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, and 7,572,631.

[00285] Generally, the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigenbinding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells, CD8+ T cells, or CD4+CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR- CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999). In some embodiments, T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS® or Trans-Act® beads, for a time period sufficient for activation of the T cells. In one aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours, e.g., 24 hours. In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gammachain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others). In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines. In some embodiments, cells activated in the presence of an anti- CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the cells are subcultured every 1, 2, 3, 4, 5, or 6 days. In some embodiments, cells are expanded for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. [00286] The expansion of T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). The expansion of T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)- 4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP or HMB-PP) or other structurally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). In some embodiments, the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-3-butenyl-l -pyrophosphate in the presence of IL-2 for one-to-two weeks. In some embodiments, the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days. In some embodiments, the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2. In some embodiments, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.

[00287] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[00288] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[00289] Once a TFP or PD-1 fusion protein is constructed, various assays known to the skilled person can be used to evaluate the activity of the molecule and/or the activity of T cells expressing the molecules, such as but not limited to, the ability of T cells to activate and expand stimulation, and anticancer activities in appropriate in vitro and animal models.

Mesothelin Associated Diseases and/or Disorders

[00290] In one aspect, the present disclosure provides methods for treating a disease associated with mesothelin expression. In one aspect, the present disclosure provides methods for treating a disease wherein part of the tumor is negative for mesothelin and part of the tumor is positive for mesothelin. For example, the modified T cell expressing an anti-mesothelin TFP and PD-1 switch receptor of the present disclosure is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of mesothelin, wherein the subject that has undergone treatment for elevated levels of mesothelin exhibits a disease associated with elevated levels of mesothelin. In one aspect, the methods provided herein are suitable for use in treating a patient having a tumor that expresses mesothelin, regardless of the intensity or level of expression. For example, in some embodiments, the present disclosure provides methods for treating a patient having a tumor wherein at least 50% of the tumor cells express mesothelin, regardless of the intensity or level of expression of mesothelin. For example, in some embodiments, the present disclosure provides methods for treating a patient having a tumor wherein at least 50% of the tumor cells are positive for mesothelin expression as measured by immunohistochemistry, wherein the positive mesothelin expression is 1+, 2+, and/or 3+ as measured by immunohistochemistry.

[00291] In one aspect, the present disclosure pertains to a method of inhibiting growth of a mesothelin- expressing tumor cell, comprising contacting the tumor cell with an engineered T cell of the present invention (e.g., a transduced T cell expressing an anti-MSLN TFP and a PD-1 switch receptor) such that the engineered T cell is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.

[00292] In one aspect, the present disclosure pertains to a method of treating cancer in a subject. The method comprises administering to the subject an engineered T cell of the present invention such that the cancer is treated in the subject. An example of a cancer that is treatable by the engineered T cell of the present disclosure is a cancer associated with expression of mesothelin. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is selected from malignant pleural mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, triple negative breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), and cholangiocarcinoma. In some embodiments, the cancer is selected from malignant pleural mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, triple negative breast cancer and colorectal cancer.

[00293] The present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and a PD-1 switch receptor; and the TFP- and PD-1 switch receptorexpressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, TFP-expressing T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. In some embodiments, without wishing to be bound by theory, the presence of the PD-1 switch receptor enhances the expansion, persistence, and/or in vivo activity of the T cells, compared to T cells that express the TFP but do not express the PD-1 switch receptor. In various aspects, the T cells administered to the patient, or their progeny, persist in the patient for at least one month, two month, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient. [00294] The present disclosure also includes a type of cellular therapy where T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a TFP and PD-1 switch receptor; and the TFP- and PD-1 switch receptor-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the T cells administered to the patient, is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.

[00295] Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the TFP- and PD-1 switch receptor-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. The transduced T cells may exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the mesothelin antigen, resist soluble mesothelin inhibition, mediate bystander killing and/or mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of mesothelin-expressing tumor may be susceptible to indirect destruction by mesothelin-redirected T cells that has previously reacted against adjacent antigenpositive cancer cells. [00296] The human modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal, e.g., a human.

[00297] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP and a PD-1 switch receptor to the cells or iii) cry opreservation of the cells.

[00298] Ex vivo procedures are well known in the art and are discussed more fully herein. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (e.g., transduced or transfected in vitro with a vector expressing a TFP disclosed herein and a PD-1 switch receptor disclosed herein. The modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[00299] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art, therefore the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL- 3 and c-kit ligand, can be used for culturing and expansion of the cells.

[00300] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[00301] Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the modified T cells of the present disclosure are used in the treatment of diseases, disorders and conditions associated with expression of mesothelin. The cells of the present disclosure may be used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of mesothelin. Thus, the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of mesothelin comprising administering to a subject in need thereof, a therapeutically effective amount of the modified T cells of the disclosure.

[00302] The modified T cells of the present disclosure may be used to treat a proliferative disease such as a cancer or malignancy or a precancerous condition. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is selected from malignant pleural mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, triple negative breast cancer, colorectal cancer, nonsmall cell lung cancer (NSCLC), or cholangiocarcinoma. Further, a disease associated with mesothelin expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing mesothelin. Non-cancer related indications associated with expression of mesothelin include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.

[00303] The modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

[00304] The present disclosure also provides methods for inhibiting the proliferation or reducing a mesothelin-expressing cell population, the methods comprising contacting a population of cells comprising a mesothelin-expressing cell with a modified T cell of the present disclosure that binds to the mesothelin-expressing cell. The anti-mesothelin modified T cell of the present disclosure may reduce the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model a cancer associated with mesothelin-expressing cells relative to a negative control. In one aspect, the subject is a human.

[00305] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with mesothelin-expressing cells e.g., a cancer expressing mesothelin), the methods comprising administering to a subject in need an anti-mesothelin modified T cell of the present disclosure that binds to the mesothelin-expressing cell. In one aspect, the subject is a human. Nonlimiting examples of disorders associated with mesothelin-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, or endometrial cancer, or atypical cancers expressing mesothelin).

[00306] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with mesothelin-expressing cells, the methods comprising administering to a subject in need an anti-mesothelin modified T cell of the present disclosure that binds to the mesothelin- expressing cell. In one aspect, the subject is a human.

[00307] The present disclosure provides methods for preventing relapse of cancer associated with mesothelin-expressing cells, the methods comprising administering to a subject in need thereof an anti- mesothelin modified T cell of the present disclosure that binds to the mesothelin-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of an anti-mesothelin modified T cell described herein that binds to the mesothelin-expressing cell in combination with an effective amount of another therapy.

Combination Therapies

[00308] In some embodiments, the TFP T cells provided herein are administered with at least one additional therapeutic agent or therapy, and/or may be used in combination with at least one additional therapeutic agent or therapy. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder. For example, the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. The delivery of one treatment can still be occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". Alternatively, the delivery of one treatment may end before the delivery of the other treatment begins, which is sometimes referred to as “sequential delivery”.

[00309] The "at least one additional therapeutic agent" may include a TFP-expressing cell. Also provided are T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen. Also provided are populations of T cells in which a first subset of T cells expresses a first TFP and a second subset of T cells express a second TFP. A TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[00310] A modified cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation. A modified cell described herein may also be used in combination with a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[00311] The subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a modified T cell provided herein. Side effects associated with the administration of a TFP-expressing cell include, but are not limited to cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. Accordingly, the methods described herein can comprise administering a modified T cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell. The soluble factor elevated in the subject is one or more of IFN-y, TNFa, IL-2, IL-6 and IL8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. Such agents include, but are not limited to, a steroid, an inhibitor of TNFa, and an inhibitor of IL-6. An example of a TNFa inhibitor is entanercept. An example of an IL-6 inhibitor is tocilizumab (toe).

[00312] Suitable additional therapeutic agents may be administered with a TFP T cell provided herein. In some aspects, the additional therapeutic agent is selected from radiation, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent, an EGFR inhibitor, an immunostimulatory agent, an anti -angiogenic agent, and combinations thereof.

[00313] Further examples of additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine). In some embodiments, the additional therapeutic agent is folinic acid, 5-fluorouracil, and/or oxaliplatin. In some embodiments, the additional therapeutic agent is 5-fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or MEK-targeted agent.

[00314] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.

[00315] In embodiments where an antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.

Pharmaceutical Compositions

[00316] Pharmaceutical compositions of the present disclosure may comprise a TFP- and PD-1 switch receptor-expressing cell, e.g., a plurality of TFP- and PD-1 switch receptor-expressing cells, as described herein (e.g., TC-510), in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure can be formulated for intravenous administration.

[00317] In some embodiments, the present disclosure provides a composition comprising transduced cells provided herein (e.g., TC-510), PlasmaLyte A, Cryostor 10, and HSA. In some embodiments, the composition comprises transduced cells provided herein (e.g., TC-510) and about 1% human serum albumin (HSA). In some embodiments the composition comprises transduced cells provided herein (e.g., TC-510), about 49% PlasmaLyte A, about 50% Cryostor 10, and about 1% HSA.

[00318] The pharmaceutical composition can be substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. The bacterium may be at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[00319] It may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present disclosure, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. T cells can be activated from blood draws of from 10 cc to 400 cc. T cells can be activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[00320] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. The T cell compositions of the present disclosure can be administered to a patient by intradermal or subcutaneous injection. The T cell compositions of the present disclosure can be administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, described herein are compositions for parenteral administration which comprises a solution of cells is dissolved or suspended in an acceptable carrier, for example, an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[00321] The TFP can be introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the disclosure, and one or more subsequent administrations of the TFP T cells of the disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. More than one administration of the TFP T cells of the present disclosure may be administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week. The subject (e.g., human subject) may receive more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject. The subject (e.g., human subject) may receive more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. The TFP T cells can be administered every other day for 3 administrations per week. The TFP T cells of the present disclosure can be administered for at least two, three, four, five, six, seven, eight or more weeks.

[00322] Mesothelin TFP T cells can be generated using lentiviral viral vectors, such as lentivirus. TFP- T cells generated that way will have stable TFP expression. TFP T cells may transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of TFPs can be affected by RNA TFP vector delivery. The TFP RNA can be transduced into the T cell by electroporation. A potential issue that can arise in patients being treated using transiently expressing TFP T cells (particularly with murine scFv bearing TFP T cells) is anaphylaxis after multiple treatments. Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-TFP response, e.g., anti-TFP antibodies having an anti- IgE isotype. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen-day break in exposure to antigen. If a patient is at high risk of generating an anti-TFP antibody response during the course of transient TFP therapy (such as those generated by RNA transductions), TFP T cell infusion breaks should not last more than ten to fourteen days.

Methods of Treatment

[00323] The engineered T cells provided herein may be useful for the treatment of any disease or condition involving mesothelin over-expression. In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a solid tumor. In some embodiments, the disease or condition is a metastatic or unresectable tumor. In some embodiments, the disease or condition is a mesothelin-expressing cancer such as MPM, ovarian adenocarcinoma (e.g., serous ovarian, fallopian tube, or primary peritoneal cancer), pancreatic cancer, triple negative breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), epithelioid MPM, or cholangiocarcinoma.

[00324] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of an engineered T cell provided herein to the subject. In some aspects, the disease or condition is a mesothelin-expressing cancer.

[00325] In some embodiments, the methods of treatment provided herein comprise administering to a subject in need thereof the engineered T cell (also referred to herein as transduced T cells) provided herein by intravenous infusion. In some embodiments, the engineered T cells are administered as one, two, three, four, five, or more doses. In some embodiments, a second dose of the engineered T cells is administered 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months following a first dose of the engineered T cells. In some embodiments, the second dose is administered between 4 months and 12 months after the first dose. In some embodiments, a dose of the engineered T cells may be administered in two, three, four, five, or more portions. In some embodiments, a first portion may comprise 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more of the dose. In some embodiments, a first portion may comprise between 25-75% of the dose. In some embodiments, a first portion may comprise 33% of the dose and a second portion may comprise 67% of the dose. In some embodiments, a first portion may comprise 50% of the dose and a second position may comprise 50% of the dose. In some embodiments, the second portion of the dose is administered 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the first portion. In some embodiments, the second portion of the dose is administered between 7-10 days after the first portion.

[00326] In some embodiments, the method of treatment provided herein further comprises retreatment of the human subject after a first administration of the engineered T cells. In some embodiments, the retreatment comprises administering a lymphodepleting chemotherapy regimen followed by a second dose of the engineered T cells. In some embodiments, the second dose of the engineered T cells may be administered as one portion, two portions, three portions, four portions, five portions, or more. In some embodiments, a first portion may comprise 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more of the dose. In some embodiments, a first portion may comprise between 25-75% of the dose. In some embodiments, a first portion may comprise 33% of the dose and a second portion may comprise 67% of the dose. In some embodiments, a first portion may comprise 50% of the dose and a second position may comprise 50% of the dose. In some embodiments, the second portion of the dose is administered 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after the first portion. In some embodiments, the second portion of the dose is administered between 7-10 days after the first portion. [00327] In some embodiments, the human subject exhibited relapse of the disease following initial treatment of the engineered T cells. In some embodiments, the subject exhibited an objective response to the initial treatment and/or showed signs and symptoms of progression prior to relapse of the disease. In some embodiments, the subject exhibited stable disease for 3, 4, 5, 6, 7, 8, or more months prior to relapse of the disease. In some embodiments, the subject exhibited a best response of stable disease, sustained for at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks following initial treatment. In some embodiments, the retreatment comprises administering a lymphodepleting chemotherapy regimen. In some embodiments, the lymphodepleting chemotherapy regimen comprises administering fludarabine at a level of 20 mg/m ² /day, 25 mg/m ² /day, 30 mg/m ² /day, 35 mg/m ² /day, 40 mg/m ² /day, 45 mg/m ² /day, or 50 mg/m ² /day on days -7 through -5 relative to administration of engineered T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administering fludarabine at a level of 30 mg/m ² /day on days -7 through -5 relative to administration of engineered T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administering cyclophosphamide at a level of 300 mg/m ² /day, 400 mg/m ² /day, 500 mg/m ² /day, 600 mg/m ² /day, 700 mg/m ² /day, or 800 mg/m ² /day on days -6 through -5 relative to administration of the engineered T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administering cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -5 relative to administration of the engineered T cells.

[00328] In some embodiments, the retreatment is administered to the human subject 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, or 14 months following completion of the initial administration of the engineered T cells. In some embodiments, the retreatment is administered to the human subject no sooner than 8 months following completion of the initial administration of the engineered T cells. In some embodiments, the retreatment is administered to the human subj ect no later than 1 year (12 months) following completion of the initial administration of the engineered T cells. In some embodiments, the retreatment is administered to the human subject no sooner than 8 months and no later than 1 year (12 months) following completion of the initial administration of the engineered T cells.

[00329] In some embodiments, the methods of treatment provided herein comprise administering to a subject in need thereof the engineered T cell (also referred to herein as transduced T cells) provided herein by intravenous infusion. In some embodiments, the engineered T cells are administered in a flat dosing regimen (i.e., a defined dose of total T cells, not based on patient body weight); or are administered in a dose per mg of body weight dosing regimen. In certain embodiments, the engineered T cells are administered in a flat dosing regimen. For example, in some embodiments, the T cells are administered at a dose ranging from about 25 x 10 ⁶ to about 200 x 10 ⁵ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 25 x 10 ⁶ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 50 x 10 ⁶ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 100 x 10 ⁶ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 130 x 10 ⁶ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 160 x 10 ⁶ transduced T cells. In some embodiments, the T cells provided herein are administered to a subject in need thereof at a dose of about 200 x 10 ⁶ transduced T cells. In some embodiments, the dose of transduced T cells administered to the subject depends on the mesothelin-expressing cancer that the subject has. For example, in some embodiments, the dosing level administered to one subject having a first mesothelin- expressing cancer is different from the dosing level administered to a second subject having a second mesothelin-expressing cancer.

[00330] Provided herein are methods of treating a human subject in need of treatment for a cancer (e.g., a MSLN-expressing cancer). The method can comprise administering to the human subject one or more doses of a population of T cells, wherein a T cell of the population of T cells comprises a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) and nucleic acid comprising a sequence encoding a PD-1 switch receptor. The population of T cells can be called, e.g., anti-MSLN TFP T cells or engineered T cells or transduced T cells or TC-510. The TFP can comprise (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain; and (b) an antibody domain comprising an anti-MSLN antigen binding domain. The TCR subunit and the anti-MSLN antigen binding domain can be operatively linked. The TFP can functionally interact with an endogenous TCR complex in the T cell. The PD-1 switch receptor can comprise a PD-1 extracellular domain or portion thereof, a PD-1 transmembrane domain, and a CD28 intracellular domain. Accordingly, in some embodiments, the present disclosure provides methods of treating a human subject in need of treatment for an MSLN-expressing cancer, comprising administering to the subject TC-510.

[00331] In embodiments, the method comprises administering the transduced T cells (“TC-510”) to the human subject at a dose of between about 50 x 10 ⁶ and about 200 x 10 ⁶ transduced T cells. For example, in embodiments, the methods provided herein comprise administering the transduced T cells (“TC-510”) to the human subject at a dose of about 50 x 10 ⁶ , about 100 x 10 ⁶ , about 130 x 10 ⁶ , about 160 x 10 ⁶ , or about 200 x 10 ⁶ .

[00332] The present disclosure also provides methods of treating a mesothelin (MSLN)-expressing cancer in a human subject in need thereof comprising: (a) obtaining a population of cells from the human subject; (b) administering to the human subject one or more doses of a population of T cells transduced with a recombinant nucleic acid comprising a sequence encoding a PD-1 switch receptor comprising a PD-1 extracellular domain or portion thereof, a PD-1 transmembrane domain, and a CD28 intracellular domain; and a recombinant nucleic acid comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising: (A) a TCR subunit comprising (i) a CD3 epsilon extracellular domain, (ii) a CD3 epsilon transmembrane domain, and (iii) a CD3 epsilon intracellular domain; and (B) an antibody domain comprising an anti-MSLN antigen binding domain. In embodiments, the methods provided herein comprise administering the transduced T cells at a dose of between about 50 x 10 ⁶ and about 200 x 10 ⁶ transduced T cells. For example, in embodiments, the methods provided herein comprise administering the transduced T cells to the human subject at a dose of about 50 x 10 ⁶ , about 100 x 10 ⁶ , about 130 x 10 ⁶ , about 160 x 10 ⁶ , or about 200 x 10 ⁶ .

[00333] The method can comprise administering to the human subject an amount of transduced anti- MSLN T cell receptor fusion protein (TFP) T cells in a flat dosing regimen. The amount of anti-MSLN TFP T cell can comprise from about 25 * 10 ⁶ to about 250 * 10 ⁶ transduced cells, for example, about 25 x 10 ⁶ , about 50 x 10 ⁶ , about 75 x 10 ⁶ , about 100 x 10 ⁶ , about 120 x 10 ⁶ , about 125 x 10 ⁶ , about 130 x 10 ⁶ , about 140 x 10 ⁶ , about 150 x 10 ⁶ , about 160 x 10 ⁶ , about 175 x 10 ⁶ , about 200 x 10 ⁶ , about 225 x 10 ⁶ , or about 250 x 10 ⁶ transduced T cells.

[00334] The cancer can be a variety of cancers expressing MSLN. For example, the cancer can comprise malignant pleural mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, triple negative breast cancer, non-small cell lung cancer (NSCLC), epithelioid MPM, or cholangiocarcinoma. In some embodiments, the serous ovarian adenocarcinoma comprises serous ovarian, fallopian tube, or primary peritoneal cancer. In some embodiments, the triple negative breast cancer is ER-negative, PR-negative, and Her2 negative status breast cancer, wherein ER-negative and PR-negative status is defined as <1%; and wherein Her2 negative status is defined as 0, 1+, or 2+ by immunohistochemistry (H4C). In some embodiments, the Her2 negative status, if 2+ by H4C, is further defined as being associated with a negative in situ hybridization test (FISH, CISH, or SISH).

[00335] In some embodiments, the method further comprises selecting the human subject by an expression level of mesothelin in a tumor sample from the human subject. In some embodiments, the tumor sample has been pathologically reviewed with confirmed positive mesothelin expression on at least about 50% tumor cells that are 1+ and/or 2+ and/or 3+ by immunohistochemistry.

[00336] In some embodiments, the MSLN-expressing cancer is mesothelioma. In some embodiments, the MSLN-expressing cancer is malignant pleural mesothelioma (MPM). In some embodiments, the MSLN-expressing cancer is ovarian adenocarcinoma. In some embodiments, the MSLN-expressing cancer is pancreatic adenocarcinoma. In some embodiments, the MSLN-expressing cancer is colorectal cancer. In some embodiments, the MSLN-expressing cancer is triple negative breast cancer. In some embodiments, the MSLN-expressing cancer is non-small cell lung cancer (NSCLC). In some embodiments, the MSLN-expressing cancer is cholangiocarcinoma. In some embodiments, the MSLN-expressing cancer is epithelioid MPM. In some embodiments, the MSLN-expressing cancer is selected from the group consisting of squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and any combinations thereof. In some embodiments, the patient has had two or more lines of prior therapy for the cancer. In some embodiments, at least one of the two or more lines of prior therapy comprises surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, radiation therapy, or any combinations thereof, (e.g., a combination therapy). [00337] In some embodiments, the human subject may have previously received one or more lines of prior treatment. In some embodiments, the human subject has received at least one and no more than five systemic therapies for metastatic or unresectable disease prior to receiving the dose of transduced T cells provided herein. The cancer in the human subject may be relapsed after one or more lines of prior treatment or is refractory or resistant to one or more lines of prior treatment. In some embodiments, the human subject may have previously received two or more lines of prior treatment. In some embodiments, the MSLN-expressing cancer is a relapsed cancer after two or more lines of prior therapy or is highly refractory or highly resistant to two or more lines of prior therapy. In some embodiments, the human subject may have previously received one, two, three, four, or five lines of prior treatment, wherein at least one of the lines of prior treatment was a systemic therapy. In some embodiments, the MSLN-expressing cancer is a relapsed cancer after one, two, three, four, or five lines of prior therapy or is highly refractory or highly resistant to one, two, three, four, or five lines of prior therapy; wherein at least one of the lines of prior therapy was a systemic therapy.

[00338] In some embodiments, at least one of the lines of prior therapy comprises a systemic therapy. In some embodiments, the systemic therapy comprises a biologic or chemotherapy. In some embodiments, the biologic comprises an antibody, antibody drug conjugate (ADC), cellular therapy, peptide, polypeptide, enzyme, vaccine, oligonucleotide, oncolytic virus, polysaccharide, or gene therapy. In some embodiments, the biologic comprises an antibody or antibody drug conjugate (ADC). In some embodiments, the checkpoint inhibitor antibody comprises nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or a biosimilar thereof. In some embodiments, the biologic comprises a cellular therapy.

[00339] In some embodiments, at least one of the lines of prior therapy comprises two or more different treatment regimes. In some embodiments, the human subject received at least one line of prior therapy comprising a systemic therapy. In some embodiments, the human subject received at least one line of prior therapy comprising a chemotherapy. In some embodiments, the human subject received at least one line of prior therapy comprising a biologic. In some embodiments, the human subject received at least one line of prior therapy comprising a combination therapy including a systemic therapy. In some embodiments, the human subject received at least two lines of prior therapy comprising a systemic therapy. In some embodiments, the human subject received at least one line of prior therapy comprising a chemotherapy. In some embodiments, the human subject received at least two lines of prior therapy comprising a chemotherapy. In some embodiments, the human subject received at least one line of prior therapy comprising a biologic. In some embodiments, the human subject received at least two lines of prior therapy comprising a biologic. In some embodiments, the human subject received at least two lines of therapy prior therapy comprising combination therapy including a systemic therapy. In some embodiments, the human subject received at least one line of prior therapy comprising a chemotherapy and at least one line of prior therapy comprising a biologic. In some embodiments, the human subject received at least one line of prior therapy comprising a chemotherapy and at least one line of prior therapy comprising a combination therapy including a systemic therapy. In some embodiments, the human subject received at least one line of prior therapy comprising a biologic and at least one line of prior therapy comprising a combination therapy including a systemic therapy. In some embodiments, the human subject received at least one line of prior therapy comprising a systemic therapy and at least one line of prior therapy comprising a surgical treatment or a combination therapy comprising a surgical treatment. In some embodiments, the human subject received at least one line of prior therapy comprising a systemic therapy and at least one line of prior therapy comprising a radiation therapy or a combination therapy comprising a radiation therapy.

[00340] In some embodiments, the human subject is at risk of recurrence. In some embodiments, the human subject has a prior history of recurrence after at least one line of prior therapy. In some embodiments, the MSLN-expressing cancer is locally advanced. In some embodiments, the MSLN- expressing cancer is metastatic. In some embodiments, the MSLN-expressing cancer is refractory to at least one line of prior therapy. In some embodiments, the MSLN-expressing cancer is refractory to at least two lines of prior therapy. In some embodiments, the MSLN-expressing cancer shows less than 20% regression after the human subject has received one line of prior therapy. In some embodiments, the MSLN-expressing cancer shows less than 20% regression after the human subject has received at least two lines of prior therapy. In some embodiments, the MSLN-expressing cancer is recurrent following one line of prior therapy. In some embodiments, the MSLN-expressing cancer is recurrent following at least two lines of prior therapy.

[00341] The MSLN-expressing cancer may be a relapsed cancer after the two or more lines of prior therapy. The MSLN-expressing cancer may be highly refractory or highly resistant to the two or more lines of prior therapy. For example, the MSLN-expressing cancer can be mesothelioma (e.g., malignant pleural mesothelioma (MPM)), ovarian adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, triple negative breast cancer, non-small cell lung cancer (NSCLC), or cholangiocarcinoma. The MSLN-expressing cancer can be selected from the group consisting of squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and any combinations thereof. [00342] In some aspects, at least one of the two or more lines of prior therapy can comprise surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, radiation therapy, or any combinations thereof. Any combinations described herein can be referred to as a combination therapy. At least one of the two or more lines of prior therapy can comprise a systemic therapy. The systemic therapy can comprise a biologic or chemotherapy. The biologic can comprise an antibody, antibody drug conjugate (ADC), cellular therapy, peptide, polypeptide, enzyme, vaccine, oligonucleotide, oncolytic virus, polysaccharide, or gene therapy. The antibody or ADC can comprise a checkpoint inhibitor antibody or ADC. The checkpoint inhibitor antibody can comprise nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or a biosimilar thereof.

[00343] In some aspects, at least one or at least one of the two or more lines of prior therapy can comprise two or more different treatment regimes, e.g., different doses or dosing schedules. For example, each treatment regimen may comprise administering a different amount of TFP T cells described herein. The human subject may have received at least one line of prior therapy comprising a systemic therapy. The human subject may have received at least one line of prior therapy comprising a chemotherapy. The human subject may have received at least one line of prior therapy comprising a biologic. The human subject may have received at least one line of prior therapy comprising a combination therapy including a systemic therapy. The human subject may have received at least two lines of prior therapy comprising a systemic therapy. The human subject may have received at least two lines of prior therapy comprising a chemotherapy. The human subject may have received at least two lines of prior therapy comprising a biologic. The human subject may have received at least two lines of therapy prior therapy comprising combination therapy including a systemic therapy. The human subject may have received at least one line of prior therapy comprising a chemotherapy and at least one line of prior therapy comprising a biologic. The human subject may have received at least one line of prior therapy comprising a chemotherapy and at least one line of prior therapy comprising a combination therapy including a systemic therapy. The human subject may have received at least one line of prior therapy comprising a biologic and at least one line of prior therapy comprising a combination therapy including a systemic therapy. The human subject may have received at least one line of prior therapy comprising a systemic therapy and at least one line of prior therapy comprising a surgical treatment or a combination therapy comprising a surgical treatment. The human subject may have received at least one line of prior therapy comprising a systemic therapy and at least one line of prior therapy comprising a radiation therapy or a combination therapy comprising a radiation therapy. [00344] The human subject described herein can have a cancer, e.g., a MSLN-expressing cancer. The human subject can be at risk of recurrence. The human subject may have a prior history of recurrence after at least one line of prior therapy. The MSLN-expressing cancer can be locally advanced. The MSLN-expressing cancer can be metastatic. The MSLN-expressing cancer can be refractory to one or more lines of prior therapy. The MSLN-expressing cancer can be refractory to at least two of two or more lines of prior therapy. The MSLN-expressing cancer may show less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or less regression after the human subject has received one or more, or two or more lines of prior therapy. The MSLN-expressing cancer can be recurrent following one or more, or two or more lines of prior therapy.

[00345] In some embodiments, the one or more prior therapy is standard frontline therapy. In some embodiments, the standard frontline therapy is platinum-based chemotherapy and/or checkpoint inhibitor therapy. In some embodiments, the one or more prior therapy is oxaliplatin or irinotecanbased regimens ± bevacizumab, and/or cetuximab or panitumumab. In some embodiments, the one or more prior therapy is fluorouracil-based (e.g., Folfirinox) or gemcitabine-based (e.g., gemcitabine ± Nab-paclitaxel) regimens. In some embodiments, the one or more prior therapy is a PARP inhibitor. In some embodiments, the one or more prior therapy is atezolizumab, alone or in combination with nab-paclitaxel.

[00346] Exemplary prior lines of therapy for certain MSLN-expressing cancers are provided. In some embodiments, the subject has MPM, wherein the subject has received standard frontline therapy (e.g., platinum -based chemotherapy or checkpoint inhibitor therapy). In some embodiments, the subject has pancreatic adenocarcinoma, wherein the patient has received frontline therapy with fluorouracil-based (e.g., FOLFIRINOX) or gemcitabine-based (e.g. gemcitabine ± Nab-paclitaxel) regimens for advanced disease, or has elected not to pursue standard front-line therapy. In some embodiments, the subject has Serous Ovarian Adenocarcinoma, wherein the subject has received a platinum -based regimen. In some embodiments, the subject has Serous Ovarian Adenocarcinoma and is a carrier of a BRCA1/2 mutation, wherein the subject has received at least a PARP inhibitor. In some embodiments, the subject has advanced Triple Negative Breast Cancer with a germline mutation in BRCA1/2, wherein the subject has received at least 1 prior systemic anti-cancer therapy, including a PARP inhibitor. In some embodiments, the subject has advanced Triple Negative Breast cancer that expresses PD-L1 on immune cells within the tumor, wherein the subject has received the combination of atezolizumab with nab-paclitaxel. In some embodiments, the subject has Colorectal Cancer with wild type KRAS, and has progressed after at least one prior standard systemic anti-cancer therapy (e.g., oxaliplatin or irinotecan-based regimens ± bevacizumab), including cetuximab or panitumumab. In some embodiments, the subject has microsatellite instability -high (MSI-H) or mismatch repair deficient (dMMR) Colorectal Cancer, wherein the subject has progressed after immune checkpoint inhibitory therapy. [00347] In some embodiments, the population of T cells is administered as a single agent. In some embodiments, a target dose of the population of T cell is about 50 x 10 ⁶ . In some embodiments, a target dose of the population of T cells is about 100 x 10 ⁶ . In some embodiments, a target dose of anti-MSLN TFP T cells is about 130 x 10 ⁶ . In some embodiments, a target dose of anti-MSLN TFP T cells is about 160 x 10 ⁶ . In some embodiments, a target dose of anti-MSLN TFP T cells is about 200 x 10 ⁶ . The administered dose may be in range of ± 20%, ± 15%, ± 10%, or ± 5% of the target dose. In some embodiments, the administered dose is in range of ± 15% of the target dose.

[00348] In some embodiments, the method further comprises administering to the human subject a lymphodepleting chemotherapy regimen prior to administration of the population of engineered T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of five doses of fludarabine and three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of four doses of fludarabine and three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of four doses of fludarabine and four doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 20 mg/m ² /day on days -9 through -3 relative to administration of the population of T cells, and further comprises administration of cyclophosphamide at a level of 700 mg/m ² /day on days -7 through -4 relative to administration of the population of T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 30 mg/m ² /day on days -7 through -4 relative to administration of the population of T cells, and further comprises administration of cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -4 relative to administration of the population of T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 40 mg/m ² /day on days -6 through -4 relative to administration of the population of T cells, and further comprises administration of cyclophosphamide at a level of 400 mg/m ² /day on days -5 through -3 relative to administration of the population of T cells.

[00349] In some embodiments, the method further comprises identifying the human subject as having a MSLN-expressing cancer. In some embodiments, the method does not induce cytokine release syndrome (CRS) above grade 1, above grade 2, or above grade 3.

[00350] Known adverse events after CAR T cell therapy include, but are not limited to, cytokine release syndrome (CRS), severe CRS (sCRS), CAR T cell-related encephalopathy syndrome (CRES) (more recently termed immune effector cell-associated neurotoxicity syndrome (ICANS)), Central Nervous System (CNS) toxicity, tumor lysis syndrome (TLS), infusion reactions, cytopenias, cardiac toxicity, hypogammaglobulinemia and graft-versus-host-disease (see, e.g., Yanez et al., CAR T Cell Toxicity: Current Management and Future Directions. HemaSphere, 2019;3:2). Non-limiting examples of CRS symptoms can include back pain, skin rash, dizziness, chills, shortness of breath, chest pain, and neurologic events. Signs of CRS can include, but are not limited to, tachycardia and hypotension. Other adverse effects of CAR T cell therapy include, but are not limited to, fever, hemodynamic changes, dyspnea and/or hypoxia and neurologic symptoms. Clinically, CRS can present with a variety of symptoms ranging from a prodromal syndrome to life-threatening manifestations. The prodromal syndrome of CRS includes a flu-like syndrome with fever, fatigue, headache, arthralgia, myalgia, and malaise. Pyrexia (fever > 38°C) is the most frequent, and usually the first, clinical sign of CRS. In some cases, it rises above 40°C and, compared to patients with mild or moderate CRS, fever in patients with sCRS peaks earlier and has a longer duration. Gastrointestinal symptoms such as nausea, diarrhea and vomiting, are also common. Severe CRS (sCRS), characterized by hemodynamic instability and organ dysfunction, is often preceded by mild or moderate signs such as hypoxia and mild hypotension. The clinical features of neurotoxicity associated with CAR T are numerous and may vary from headache, pain, memory loss, meningismus, dizziness, alterations in mental status (somnolence, disorientation, impaired attention, agitation, delirium, coma), movement disorders (tremor, myoclonus, facial automatisms), impaired speech (dysartria, aphasia), seizures and encephalopathy to coma.

[00351] In various embodiments, the methods, provided herein can further comprise selecting the human subject for the treatment provided herein. The selection of the human subject can be determined by an expression level of mesothelin in a tumor sample from the human subject. The expression of mesothelin can be determined by various methods, for example, immunohistochemistry. For example, the tumor sample can be pathologically reviewed with confirmed positive mesothelin expression on at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more tumor cells that are 1+, 2+, and/or 3+ by immunohistochemistry. In some embodiments, the tumor sample has been pathologically reviewed with confirmed positive mesothelin expression on at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more tumor cells that are 1+, 2+, and/or 3+ by immunohistochemistry. In some embodiments, the tumor sample has been pathologically reviewed with confirmed positive mesothelin expression on at least about 50% of the tumor cells, wherein the intensity of mesothelin expression is 1+, 2+, and/or 3+ on at least about 50% of the tumor cells.

[00352] In some embodiments, administration of the population of T cells results in at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 70% regression in tumor volume. In some embodiments, the regression in tumor volume is maintained for at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.

[00353] In some embodiments, the population of T cells is administered at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months following one or more prior lines of therapy.

[00354] The method can further comprise administering to the human subject a lymphodepleting chemotherapy regimen prior to administration of the first portion of a dose of the population of T cells. [00355] The lymphodepleting chemotherapy regimen can comprise administration of at least one dose of fludarabine and at least one dose of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise administration of five doses of fludarabine and three doses of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise administration of four doses of fludarabine and three doses of cyclophosphamide. The lymphodepleting chemotherapy regimen can comprise administration of four doses of fludarabine and four doses of cyclophosphamide. [00356] The lymphodepleting chemotherapy regimen can comprise any dosing schedule of fludarabine or cyclophosphamide described herein. For example, the lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 20 mg/m ² /day on days -9 through -3 relative to administration of the population of T cells. The lymphodepleting chemotherapy regimen can further comprise administration of cyclophosphamide at a level of about 700 mg/m ² /day on days -7 through -4 relative to administration of the population of T cells. The lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 30 mg/m ² /day on days -7 through -4 relative to administration of the population of T cells. The lymphodepleting chemotherapy regimen can further comprise administration of cyclophosphamide at a level of about 600 mg/m ² /day on days -6 through -4 relative to administration of the population of T cells.

[00357] The lymphodepleting chemotherapy regimen can comprise administration of fludarabine at a level of about 40 mg/m ² /day on days -6 through -4 relative to administration of the population of T cells. The lymphodepleting chemotherapy regimen can comprise administration of cyclophosphamide at a level of about 400 mg/m ² /day on days -5 through -3 relative to administration of the population of T cells.

[00358] The method can further comprise obtaining a population of cells from the human subject prior to administration of the one or more doses of the population of T cells. In some embodiments, the population of cells is obtained from the human subject prior to initiation of the lymphodepleting chemotherapy regimen. The method can further comprise transducing T cells from the population of cells with a recombinant nucleic acid comprising a sequence encoding the TFP and a sequence encoding the PD-1 switch receptor, thereby generating the population of T cells (e.g., TC-510). The method can further comprise identifying the human subject as having a MSLN-expressing cancer. The method may not induce cytokine release syndrome (CRS). For example, the method may not induce CRS above grade 1, above grade 2, or above grade 3.

[00359] Also provided herein is a method of treating a mesothelin (MSLN)-expressing cancer in a human subject in need thereof after one, two, three, four, or five lines of prior therapy for treating the MSLN-expressing cancer. The method can comprise (a) obtaining a population of cells from the human subject; and (b) administering to the human subject one or more doses of a population of T cells transduced with a recombinant nucleic acid described herein comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a PD-1 fusion protein. The TFP can comprise (I) a TCR subunit comprising (i) a CD3 epsilon extracellular domain, (ii) a CD3 epsilon transmembrane domain, and (iii) a CD3 epsilon intracellular domain; and (II) an antibody domain comprising an anti- MSLN antigen binding domain. The TCR subunit and the antibody domain can be operatively linked. The TFP can be capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. The PD-1 fusion protein can comprise a PD-1 extracellular domain or portion thereof, a PD-1 transmembrane domain, and a CD28 intracellular domain. The population of cells can be TC-510, or a composition comprising TC-510. The human subject may have previously received the prior lines of prior therapy for treating the MSLN-expressing cancer. The MSLN- expressing cancer may be relapsed after the lines of prior therapy, or may be highly refractory or highly resistant to the lines of prior therapy.

[00360] The population of cells obtained from the human subject can be PBMCs. The population of T cells can comprise a population of CD4+ and CD8+ T cells isolated from the PBMCs prior to transduction with the recombinant nucleic acid. The human subject may have previously been identified as having a MSLN-expressing cancer. The human subject can be identified as having a MSLN-expressing cancer by immunohistochemistry and/or flow cytometry to identify MSLN expression on cancerous cells from the human subject. The human subject may have been diagnosed with the MSLN-expressing cancer.

[00361] Also provided herein are methods of treating a subject with a disease, disorder or condition. A method of treatment can comprise administering a pharmaceutical composition disclosed herein to a subject with a disease, disorder or condition. The present disclosure provides methods of treatment comprising an immunogenic therapy. Methods of treatment for a disease (such as cancer or a viral infection) are provided. A method can comprise administering to a subject an effective amount of a pharmaceutical composition comprising the transduced T cells.

[00362] In some embodiments, the method of treating a subject with a disease or condition comprises administering to the subject the pharmaceutical composition disclosed herein. In some embodiments, the method is a method of preventing resistance to a cancer therapy, wherein the method comprises administering to a subject in need thereof the pharmaceutical composition disclosed herein. In some embodiments, the method is a method of inducing an immune response, wherein the method comprises administering to a subject in need thereof the pharmaceutical composition disclosed herein. In some embodiments, the immune response is a humoral response. In some embodiments, the immune response is a cytotoxic T cell response.

[00363] In some embodiments, the subject has cancer, wherein the cancer is selected from the group consisting of mesothelioma, ovarian cancer, triple-negative breast cancer, colorectal cancer, pancreatic adenocarcinoma, cholangiocarcinoma, and lung adenocarcinoma.

[00364] In some embodiments, the method further comprises administering at least one additional therapeutic agent or modality. In some embodiments, the at least one additional therapeutic agent or modality is surgery, a checkpoint inhibitor, an antibody or fragment thereof, a chemotherapeutic agent, radiation, a vaccine, a small molecule, a T cell, a vector, and APC, a polynucleotide, an oncolytic virus or any combination thereof. In some embodiments, the at least one additional therapeutic agent is an anti-PD-1 agent and anti-PD-Ll agent, an anti-CTLA-4 agent, or an anti-CD40 agent. In some embodiments, the additional therapeutic agent is administered before, simultaneously, or after administering the pharmaceutical composition disclosed herein.

[00365] Additionally, the disease or condition provided herein includes refractory or recurrent malignancies whose growth may be inhibited using the methods of treatment of the present disclosure. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, stomach cancer, bladder cancer, gall bladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, squamous cell carcinoma of the head and neck, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematological cancer, leukemia, lymphoma, neuroma, and combinations thereof. In some embodiments, a cancer to be treated by the methods of the present disclosure include, for example, carcinoma, squamous carcinoma (for example, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder, tongue, larynx, and gullet), and adenocarcinoma (for example, prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, gullet, rectum, uterus, stomach, mammary gland, and ovary). In some embodiments, a cancer to be treated by the methods of the present disclosure further include sarcomata (for example, myogenic sarcoma), leukosis, neuroma, melanoma, and lymphoma. In some embodiments, a cancer to be treated by the methods of the present disclosure is breast cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is triple negative breast cancer (TNBC). In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is ovarian cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is colorectal cancer. In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is non-small cell lung cancer (NSCLC). In some embodiments, a cancer to be treated by the methods of treatment of the present disclosure is cholangiocarcinoma.

[00366] In some embodiments, a patient or population of patients to be treated with a pharmaceutical composition of the present disclosure have a solid tumor. In some embodiments, a solid tumor is a melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma. Specific examples of cancers that can be prevented and/or treated in accordance with present disclosure include, but are not limited to, malignant pleural mesothelioma (MPM), pancreatic cancer, triple negative breast cancer, colorectal cancer, serous ovarian adenocarcinoma, NSCLC, and cholangiocarcinoma.

[00367] The pharmaceutical compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). In some embodiments, at least one or more chemotherapeutic agents may be administered in addition to the pharmaceutical composition comprising an immunogenic therapy. In some embodiments, the one or more chemotherapeutic agents may belong to different classes of chemotherapeutic agents.

[00368] In some embodiments, the methods of treatment include one or more rounds of leukapheresis prior to transplantation of T cells. The leukapheresis may include collection of peripheral blood mononuclear cells (PBMCs). Leukapheresis may include mobilizing the PBMCs prior to collection. Alternatively, non-mobilized PBMCs may be collected. A large volume of PBMCs may be collected from the subject in one round. Alternatively, the subject may undergo two or more rounds of leukapheresis. The volume of apheresis may be dependent on the number of cells required for transplant. For instance, 12-15 liters of non-mobilized PBMCs may be collected from a subject in one round. The number of PBMCs to be collected from a subject may be between IxlO ⁸ to 5xl0 ¹⁰ cells. The number of PBMCs to be collected from a subject may be IxlO ⁸ , 5xl0 ⁸ , IxlO ⁹ , 5xl0 ⁹ , IxlO ¹⁰ or 5xl0 ¹⁰ cells. The minimum number of PBMCs to be collected from a subject may be lxl0 ⁶ /kg of the subject’s weight. The minimum number of PBMCs to be collected from a subject may be lxl0 ⁶ /kg, 5xl0 ⁶ /kg, lxl0 ⁷ /kg, 5xl0 ⁷ /kg, lxl0 ⁸ /kg, 5xl0 ⁸ /kg of the subject’s weight. [00369] In some embodiments, the methods of treatment include cancer treatment of a subject prior to administering transduced T cells provided herein. The cancer treatment may include chemotherapy, immunotherapy, targeted agents, radiation and high dose corticosteroid. The methods may include administering chemotherapy to a subject including lymphodepleting chemotherapy using high doses of myeloablative agents. In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the first or subsequent dose. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, 7, 8, 9 or 10 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 10 days prior, such as no more than 9, 8, 7, 6, 5, 4, 3, or 2 days prior, to the first or subsequent dose.

[00370] In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered between 0.3 grams per square meter of the body surface of the subject (g/m ² ) and 5 g/m ² cyclophosphamide. In some cases, the amount of cyclophosphamide administered to a subject is about at least 0.3 g/m ² . In some cases, the amount of cyclophosphamide administered to a subject is about at most 5 g/m ² . In some cases, the amount of cyclophosphamide administered to a subject is about 0.3 g/m ² to 0.4 g/m ² , 0.3 g/m ² to 0.5 g/m ² , 0.3 g/m ² to 0.6 g/m ² , 0.3 g/m ² to 0.7 g/m ² , 0.3 g/m ² to 0.8 g/m ² , 0.3 g/m ² to 0.9 g/m ² , 0.3 g/m ² to 1 g/m ² , 0.3 g/m ² to 2 g/m ² , 0.3 g/m ² to 3 g/m ² , 0.3 g/m ² to 4 g/m ² , 0.3 g/m ² to 5 g/m ² , 0.4 g/m ² to 0.5 g/m ² , 0.4 g/m ² to 0.6 g/m ² , 0.4 g/m ² to 0.7 g/m ² , 0.4 g/m ² to 0.8 g/m ² , 0.4 g/m ² to 0.9 g/m ² , 0.4 g/m ² to 1 g/m ² , 0.4 g/m ² to 2 g/m ² , 0.4 g/m ² to 3 g/m ² , 0.4 g/m ² to 4 g/m ² , 0.4 g/m ² to 5 g/m ² , 0.5 g/m ² to 0.6 g/m ² , 0.5 g/m ² to 0.7 g/m ² , 0.5 g/m ² to 0.8 g/m ² , 0.5 g/m ² to 0.9 g/m ² , 0.5 g/m ² to 1 g/m ² , 0.5 g/m ² to 2 g/m ² , 0.5 g/m ² to 3 g/m ² , 0.5 g/m ² to 4 g/m ² , 0.5 g/m ² to 5 g/m ² , 0.6 g/m ² to 0.7 g/m ² , 0.6 g/m ² to 0.8 g/m ² , 0.6 g/m ² to 0.9 g/m ² , 0.6 g/m ² to 1 g/m ² , 0.6 g/m ² to 2 g/m ² , 0.6 g/m ² to 3 g/m ² , 0.6 g/m ² to 4 g/m ² , 0.6 g/m ² to 5 g/m ² , 0.7 g/m ² to 0.8 g/m ² , 0.7 g/m ² to 0.9 g/m ² , 0.7 g/m ² to 1 g/m ² , 0.7 g/m ² to 2 g/m ² , 0.7 g/m ² to 3 g/m ² , 0.7 g/m ² to 4 g/m ² , 0.7 g/m ² to 5 g/m ² , 0.8 g/m ² to 0.9 g/m ² , 0.8 g/m ² to 1 g/m ² , 0.8 g/m ² to 2 g/m ² , 0.8 g/m ² to 3 g/m ² , 0.8 g/m ² to 4 g/m ² , 0.8 g/m ² to 5 g/m ² , 0.9 g/m ² to 1 g/m ² , 0.9 g/m ² to 2 g/m ² , 0.9 g/m ² to 3 g/m ² , 0.9 g/m ² to 4 g/m ² , 0.9 g/m ² to 5 g/m ² , 1 g/m ² to 2 g/m ² , 1 g/m ² to 3 g/m ² , 1 g/m ² to 4 g/m ² , 1 g/m ² to 5 g/m ² , 2 g/m ² to 3 g/m ² , 2 g/m ² to 4 g/m ² , 2 g/m ² to 5 g/m ² , 3 g/m ² to 4 g/m ² , 3 g/m ² to 5 g/m ² , or 4 g/m ² to 5 g/m ² . In some cases, the amount of cyclophosphamide administered to a subject is about 0.3 g/m ² , 0.4 g/m ² , 0.5 g/m ² , 0.6 g/m ² , 0.7 g/m ² , 0.8 g/m ² , 0.9 g/m ² , 1 g/m ² , 2 g/m ² , 3 g/m ² , 4 g/m ² , or 5 g/m ² . In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 200 mg/kg and 1000 mg/kg, such as between or between about 400 mg/kg and 800 mg/kg. In some aspects, the subject is preconditioned with or with about 600 mg/kg of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. For example, in some instances, the agent, e.g., cyclophosphamide, is administered between or between about 1 and 5 times, such as between or between about 2 and 4 times. In some embodiments, such plurality of doses is daily, such as on days -6 through -4 relative to administration of anti-MSLN TFP T cells.

[00371] In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 milligrams per square meter of the body surface of the subject (mg/m ² ) and 100 mg/m ² . In some cases, the amount of fludarabine administered to a subject is about at least 1 mg/m ² . In some cases, the amount of fludarabine administered to a subject is about at most 100 mg/m ² . In some cases, the amount of fludarabine administered to a subject is about 1 mg/m ² to 5 mg/m ² , 1 mg/m ² to 10 mg/m ² , 1 mg/m ² to 15 mg/m ² , 1 mg/m ² to 20 mg/m ² , 1 mg/m ² to 30 mg/m ² , 1 mg/m ² to 40 mg/m ² , 1 mg/m ² to 50 mg/m ² , 1 mg/m ² to 70 mg/m ² , 1 mg/m ² to 90 mg/m ² , 1 mg/m ² to 100 mg/m ² , 5 mg/m ² to 10 mg/m ² , 5 mg/m ² to 15 mg/m ² , 5 mg/m ² to 20 mg/m ² , 5 mg/m ² to 30 mg/m ² , 5 mg/m ² to 40 mg/m ² , 5 mg/m ² to 50 mg/m ² , 5 mg/m ² to 70 mg/m ² , 5 mg/m ² to 90 mg/m ² , 5 mg/m ² to 100 mg/m ² , 10 mg/m ² to 15 mg/m ² , 10 mg/m ² to 20 mg/m ² , 10 mg/m ² to 30 mg/m ² , 10 mg/m ² to 40 mg/m ² , 10 mg/m ² to 50 mg/m ² , 10 mg/m ² to 70 mg/m ² , 10 mg/m ² to 90 mg/m ² , 10 mg/m ² to 100 mg/m ² , 15 mg/m ² to 20 mg/m ² , 15 mg/m ² to 30 mg/m ² , 15 mg/m ² to 40 mg/m ² , 15 mg/m ² to 50 mg/m ² , 15 mg/m ² to 70 mg/m ² , 15 mg/m ² to 90 mg/m ² , 15 mg/m ² to 100 mg/m ² , 20 mg/m ² to 30 mg/m ² , 20 mg/m ² to 40 mg/m ² , 20 mg/m ² to 50 mg/m ² , 20 mg/m ² to 70 mg/m ² , 20 mg/m ² to 90 mg/m ² , 20 mg/m ² to 100 mg/m ² , 30 mg/m ² to 40 mg/m ² , 30 mg/m ² to 50 mg/m ² , 30 mg/m ² to 70 mg/m ² , 30 mg/m ² to 90 mg/m ² , 30 mg/m ² to 100 mg/m ² , 40 mg/m ² to 50 mg/m ² , 40 mg/m ² to 70 mg/m ² , 40 mg/m ² to 90 mg/m ² , 40 mg/m ² to 100 mg/m ² , 50 mg/m ² to 70 mg/m ² , 50 mg/m ² to 90 mg/m ² , 50 mg/m ² to 100 mg/m ² , 70 mg/m ² to 90 mg/m ² , 70 mg/m ² to 100 mg/m ² , or 90 mg/m ² to 100 mg/m ² . In some cases, the amount of fludarabine administered to a subject is about 1 mg/m ² , 5 mg/m ² , 10 mg/m ² , 15 mg/m ² , 20 mg/m ² , 30 mg/m ² , 40 mg/m ² , 50 mg/m ² , 70 mg/m ² , 90 mg/m ² , or 100 mg/m ² . In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. For example, in some instances, the agent, e.g., fludarabine, is administered between or between about 1 and 5 times, such as between or between about 3 and 5 times. In some embodiments, such plurality of doses is administered daily, such as on days -7 through -4 relative to administration of anti-MSLN TFP T cells.

[00372] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 400 mg/m ² of cyclophosphamide and one or more doses of 20 mg/m ² fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 500 mg/m ² of cyclophosphamide and one or more doses of 25 mg/m ² fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 600 mg/m ² of cyclophosphamide and one or more doses of 30 mg/m ² fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 700 mg/m ² of cyclophosphamide and one or more doses of 35 mg/m ² fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 700 mg/m ² of cyclophosphamide and one or more doses of 40 mg/m ² fludarabine prior to the first or subsequent dose of T cells. In some examples, the subject is administered 800 mg/m ² of cyclophosphamide and one or more doses of 45 mg/m ² fludarabine prior to the first or subsequent dose of T cells.

[00373] Fludarabine and cyclophosphamide may be administered on alternative days. In some cases, fludarabine and cyclophosphamide may be administered concurrently. In some cases, an initial dose of fludarabine is followed by a dose of cyclophosphamide. In some cases, an initial dose of cyclophosphamide may be followed by an initial dose of fludarabine. In some examples, a treatment regimen may include treatment of a subject with an initial dose of fludarabine 10 days prior to the transplant, followed by treatment with an initial dose of cyclophosphamide administered 9 days prior to the cell transplant, concurrently with a second portion of a dose of fludarabine. In some examples, a treatment regimen may include treatment of a subject with an initial dose of fludarabine 8 days prior to the transplant, followed by treatment with an initial dose of cyclophosphamide administered 7 days prior to the transplant concurrently with a second portion of a dose of fludarabine.

[00374] The transduced T cell product (e.g., TC-510) may be administered as one or more infusions. In some cases, a subject is administered one dose of T cells. In some cases, a subject is administered more than one doses of T cells. In some cases, a subject is administered three doses of T cells. In some cases, a subject is administered four doses of T cells. In some cases, a subject is administered five or more doses of T cells. In some embodiments, two consecutive doses of T cells are administered no less than 4 months and no more than 12 months apart. In some embodiments, a second dose of T cells is administered no sooner than 4 months and no later than 12 months (1 year; 52 weeks) following completion of the first dose of T cells. In some embodiments, the more than one doses of T cells are evenly spaced. In some embodiments, the one or more doses of T cells are not evenly spaced.

[00375] A single infusion may comprise a dose between 25 x 10 ⁶ transduced cells and 250 x 10 ⁶ transduced cells. A single infusion may comprise between about 50 x 10 ⁶ transduced cells and about 200 x 10 ⁶ transduced cells. A single infusion may comprise about 25 xlO ⁶ transduced cells, about 50 x 10 ⁶ transduced cells, about 100 x 10 ⁶ transduced cells, about 130 x 10 ⁶ transduced cells, about 160 x 10 ⁶ transduced cells, or about 200 x 10 ⁶ transduced cells.

[00376] For example, a subject can be administered a dose of about 50xl0 ⁶ transduced cells. For example, a subject that has undergone a lymphodepletion treatment can be administered a dose of about 5 Ox 10 ⁶ transduced cells.

[00377] For example, a subject can be administered a dose of about lOOxlO ⁶ transduced cells. For example, a subject that has undergone a lymphodepletion treatment can be administered a dose of about lOOxlO ⁶ transduced cells.

[00378] For example, a subject can be administered a dose of about 13 Ox 10 ⁶ transduced cells. For example, a subject that has undergone a lymphodepletion treatment can be administered a dose of about 13 Ox 10 ⁶ transduced cells.

[00379] For example, a subject can be administered a dose of about 160xl0 ⁶ transduced cells. For example, a subject that has undergone a lymphodepletion treatment can be administered a dose of about 160xl0 ⁶ transduced cells.

[00380] For example, a subject can be administered a dose of about 200xl0 ⁶ transduced cells. For example, a subject that has undergone a lymphodepletion treatment can be administered a dose of about 200xl0 ⁶ transduced cells.

[00381] In some embodiments, a subject is administered more than one dose of T cells and each dose has the same number of transduced T cells. In some embodiments, a subject is administered more than one dose of T cells and one or more of the doses do not have the same number of transduced T cells.

[00382] In one example, the method of treatment may comprise an initial PBMC collection from a subject. IxlO ⁶ to IxlO ⁸ PBMCs/kg of the subject weight may be collected. The PBMC fraction collected from the subject may then be enriched for T cells. Enriched T cells may be transduced as described herein to express an anti-MSLN T cell receptor fusion protein (TFP) and a PD-1 fusion protein. In some cases, the transduced T cells may be expanded and/or cryopreserved. The subject may undergo lymphodepleting chemotherapy following the leukapheresis. An alternating dose of fludarabine and cyclophosphamide may be administered to the subject. The dosing schedule may be one described elsewhere herein. In one example, the dose of fludarabine or an equivalent chemotherapeutic agent administered to the subject may be between 15 mg/m ² to 45 mg/m ² . The dose of cyclophosphamide or an equivalent chemotherapeutic agent administered to the subject may be between 400g/m ² to 800 mg/m ² . The doses of fludarabine and cyclophosphamide may be administered in an alternating manner, for instance in this scenario an initial dose of fludarabine may be followed by an initial dose of cyclophosphamide. The administration of lymphodepleting agents may be followed by the transplantation of anti-MSLN TFP producing T cells. T cells may be administered intravenously as a single dose to the subject. A single infusion of cells may comprise between 25xl0 ⁶ and 200 x 10 ⁶ transduced T cells.

[00383] In another example, the method of treatment may comprise an initial PBMC collection from a subject. IxlO ⁶ to IxlO ⁸ PBMCs/kg of the subject weight may be collected. The PBMC fraction collected from the subject may then be enriched for T cells. Enriched T cells may be transduced as described herein to express anti-MSLN T cell receptor fusion protein (TFP). An alternating dose of fludarabine and cyclophosphamide may be administered to the subject after the leukapheresis is complete. The dosing schedule may be one described elsewhere herein. In one example, the dose of fludarabine or an equivalent chemotherapeutic agent administered to the subject may be 20 mg/m ² . The dose of cyclophosphamide or an equivalent chemotherapeutic agent administered to the subject may be 700g/m ² . The doses of fludarabine and cyclophosphamide may be administered in an alternating manner, for instance in this scenario an initial dose of fludarabine may be followed by an initial dose of cyclophosphamide. An initial dose of fludarabine may be administered on day -9 of the T cell transplant. Other doses of fludarabine may be administered on days -8, -7, -6, -5, -4 and -3. An initial dose of cyclophosphamide may be administered on day -7 concurrently with the fludarabine. Other doses of cyclophosphamide may be administered on days -5 and -4. The administration of lymphodepleting agents may be followed by the transplantation of anti-MSLN TFP producing T cells. T cells may be administered intravenously as a single dose to the subject. A single infusion of cells may comprise between 25xl0 ⁶ and 200 x 10 ⁶ transduced T cells.

[00384] In another example, the method of treatment may comprise an initial PBMC collection from a subject. IxlO ⁶ to IxlO ⁸ PBMCs/kg of the subject weight may be collected. The PBMC fraction collected from the subject may then be enriched for T cells. Enriched T cells may be transduced as described herein to express anti-MSLN T cell receptor fusion protein (TFP). An alternating dose of fludarabine and cyclophosphamide may be administered to the subject after the leukapheresis is complete. The dosing schedule may be one described elsewhere herein. In one example, the dose of fludarabine or an equivalent chemotherapeutic agent administered to the subject may be 40 mg/m ² . The dose of cyclophosphamide or an equivalent chemotherapeutic agent administered to the subject may be 400g/m ² . The doses of fludarabine and cyclophosphamide may be administered in an alternating manner, for instance in this scenario an initial dose of fludarabine may be followed by an initial dose of cyclophosphamide. An initial dose of fludarabine may be administered on day -6 of the T cell transplant. Other doses of fludarabine may be administered on days -5 and -4. An initial dose of cyclophosphamide may be administered on day -5 concurrently with the fludarabine. Another dose of cyclophosphamide may be administered on day -3. The administration of lymphodepleting agents may be followed by the transplantation of anti-MSLN TFP producing T cells. T cells may be administered intravenously as a single dose to the subject. A single infusion of cells may comprise between 25xl0 ⁶ and 200 x 10 ⁶ transduced T cells.

[00385] In another example, the method of treatment may comprise an initial PBMC collection from a subject. IxlO ⁶ to IxlO ⁸ PBMCs/kg of the subject weight may be collected. The PBMC fraction collected from the subject may then be enriched for T cells. Enriched T cells may be transduced as described herein to express anti-MSLN T cell receptor fusion protein (TFP). In some cases, the transduced T cells may be expanded and/or cryopreserved. In this example, the subject does not undergo lymphodepleting chemotherapy following the leukapheresis. Anti-MSLN TFP producing T cells may be administered intravenously as a single dose to the subject. A single infusion of cells may comprise between 25xl0 ⁶ and 200 x 10 ⁶ transduced T cells.

[00386] In some embodiments, the dosing of the transduced cells is fractionated such that the subject is administered the dose in a first portion and a second portion of the dose. In some embodiments, the subject is administered the dose in a first portion and two or more additional portions of the dose (e.g., a second portion and a third portion, or more). In some embodiments, the first portion of a dose comprise a first number of transduced cells and wherein the second portion of a dose comprise a second number of transduced cells, wherein the ratio of the first number of transduced cells to the second number of transduced cellsis from 1 : 10 to 10: 1 For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7:1, 8: 1 or 9: 1. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 1 :9.5, 1 :8.5, 1 :7.5, 1 :6.5, 1 :5.5, 1 :4.5, 1 :3.5, 1 :2.5, 1 : 1.5, 2.5: 1, 3.5: 1, 4.5: 1, 5.5: 1, 6.5: 1, 7.5: 1, 8.5: 1 or 9.5: 1. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 2:9, 2:8, 2:7, 2:6, 2:5, 2:4, 2:3, 3:2, 4:2, 5:2, 6:2, 7:2, 8:2 or 9:2. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 3:8, 3:7, 3:5, 3:4, 4:3, 5:3, 7:3 or 8:3. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 4:9, 4:7, 4:5, 5:4, 7:4, or 9:4. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 5:9, 5:8, 5:7, 5:6, 6:5, 7:5, 8:5 or 9:5. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 6:7 or 7:6. For example, the ratio of the first number of transduced cells to the second number of transduced cells can be about 7:8 or 8:7. For example, the ratio of the first number of transduced cellsto the second number of transduced cells can be about 8:9 or 9:8.

[00387] In some embodiments, the ratio of the first number of transduced cellsto the second number of transduced cells is not 1 : 1. In some embodiments, the first portion of a dose and the second portion of a dose comprise from about one-third to two-thirds of a reference dose, and wherein the reference dose is about 25 x io ⁶ to about 200 x io ⁶ transduced cells, for example, about 20 xlO ⁶ , about 50 xlO ⁶ , about 100 xlO ⁶ , about 130 xlO ⁶ , about 160 xlO ⁶ , or about 200 xlO ⁶ transduced cells.

[00388] In some embodiments, the second portion of a dose is administered less than 50 days following administration of the first portion of a dose of transduced T cells. For example, the second portion of a dose can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 days following administration of the first portion of a dose of transduced T cells. In some embodiments, the second portion of a dose is administered about 7 days following administration of the first portion of a dose of transduced T cells.

[00389] In some embodiments, the sum of the first portion of a dose and the second portion of a dose equals a reference dose. In some embodiments, the reference dose is about 25 x io ⁶ to about 200 x io ⁶ transduced cells, for example, about 20 xlO ⁶ , about 50 xlO ⁶ , about 100 xlO ⁶ , about 130 xlO ⁶ , about 160 xlO ⁶ , or about 200 xlO ⁶ transduced cells.

[00390] In some embodiments, the first portion of a dose is about 16.66 x 10 ⁶ transduced cells. In some embodiments, the second portion of a dose is about 33.33 x io ⁶ transduced cells. In some embodiments, the first portion of a dose is about 33.33x 10 ⁶ transduced cells. In some embodiments, the second portion of a dose is about 66.67 x io ⁶ transduced cells. In some embodiments, the first portion of a dose is about 43.29 x 10 ⁶ transduced cells. In some embodiments, the second portion of a dose is about 86.71 x io ⁶ transduced cells. In some embodiments, the first portion of a dose is about 53.28 x 10 ⁶ transduced cells. In some embodiments, the second portion of a dose is about 10.67 x io ⁷ transduced cells. In some embodiments, the first portion of a dose is about 66.6 x 10 ⁶ transduced cells. In some embodiments, the second portion of a dose is about 13.34 x 10 ⁷ transduced cells.

[00391] In some embodiments, the second portion of a dose is administered less than 10 days following administration of the first portion of a dose of anti-MSLN TFP T cells. In some embodiments, the second portion of a dose is administered 7 or fewer days following administration of the first portion of a dose of anti-MSLN TFP T cells. In some embodiments, the second portion of a dose is administered at least three days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered three to seven days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 3 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 4 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 5 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 6 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 7 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 8 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 9 days after administering the first portion of a dose. In some embodiments, the second portion of a dose is administered 10 days after administering the first portion of a dose.

[00392] In some embodiments, the human subject does not exhibit any adverse event after administering the first portion of a dose. In some embodiments, the human subject exhibits an adverse event after administering the first portion of a dose, and wherein the second portion of a dose is administered at least seven days after administering the first portion of a dose. In some embodiments, the adverse event is selected from the group consisting of cytokine release syndrome (CRS), neurotoxicity, severe CRS (sCRS), CAR T cell-related encephalopathy syndrome (CRES) or immune effector cell-associated neurotoxicity syndrome (ICANS), Central Nervous System (CNS) toxicity, tumor lysis syndrome (TLS), an infusion reaction, cytopenia, cardiac toxicity, hypogammaglobulinemia, graft-versus-host-disease and any combination thereof. In some embodiments, the adverse event is selected from the group consisting of cytokine release syndrome and neurotoxicity. In some embodiments, the adverse event is > grade 3 cytokine release syndrome and/or > grade 2 neurotoxicity.

[00393] In some embodiments, the human subject exhibits an adverse event and the second portion of a dose is administered at a first timepoint after administration of the first portion of a dose of transduced T cells, wherein the first timepoint is longer than the second timepoint. In some embodiments, the human subject exhibits an adverse event that is grade 1 or lower and the second portion of a dose is administered at a first timepoint after administration of the first portion of a dose of transduced T cells. In some embodiments, the human subject exhibits an adverse event that is grade 1 or higher and the second portion of a dose is administered at a first timepoint after administration of the first portion of a dose of transduced T cells. In some embodiments, the human subject exhibits an adverse event that is grade 2 or higher and the second portion of a dose is administered at a first timepoint after administration of the first portion of a dose of transduced T cells. In some embodiments, the human subject exhibits an adverse event that is grade 3 or higher and the second portion of a dose is administered at a first timepoint after administration of the first portion of a dose of transduced T cells. In some embodiments, the first timepoint is more than 7 days after the first portion of a dose is administered. For example, the first timepoint can be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 days after the first portion of a dose is administered. [00394] In some embodiments, the human subject does not exhibit an adverse event that is grade 2 or higher and the second portion of a dose is administered at a second timepoint after administration of the first portion of a dose of transduced T cells, wherein the first timepoint is longer than the second timepoint. In some embodiments, the human subject does not exhibit an adverse event that is grade 3 or higher and the second portion of a dose is administered at a second timepoint after administration of the first portion of a dose of transduced T cells. In some embodiments, the human subject exhibits an adverse event that is grade 1 or lower and the second portion of a dose is administered at a second timepoint after administration of the first portion of a dose of transduced T cells.

[00395] In some embodiments, the second timepoint is 7 days or less after the first portion of a dose is administered. For example, the second timepoint can be 1, 2, 3, 4, 5, 6 or 7 days after the first portion of a dose is administered. In some embodiments, the second timepoint is after the adverse event is no longer exhibited by the subject. In some embodiments, the second timepoint is after the adverse event exhibited by the subject is grade 1 or less.

[00396] In some embodiments, the method reduces or prevents a risk of adverse event associated with administering a dose with an amount equivalent to the sum of the amount of the first portion of a dose plus the second portion of a dose. In some embodiments, the adverse event comprises bacterial or fungal infection, tumor lysis syndrome, cytokine release syndrome, neurotoxicity, infusion reactions, cytopenias, cardiac toxicity, hypogammaglobulinemia or graft-versus-host disease.

[00397] In some embodiments, the human subject has or has not received one or more lines of prior therapy prior to administering the first portion of a dose. In some embodiments, the one or more lines of prior therapy comprises surgery, chemotherapy, hormonal therapy, biological therapy, antibody therapy, radiation therapy, systemic therapy, or any combinations thereof.

[00398] In some embodiments, the human subject has received a lymphodepl eting chemotherapy regimen prior to administering the first portion of a dose. In some embodiments, the human subject has not received a lymphodepl eting chemotherapy regimen prior to administering the first portion of a dose. In some embodiments, the method further comprises administering a lymphodepleting chemotherapy regimen to the human subject prior to administering the first portion of a dose. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of about four doses of fludarabine and about three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of four doses of fludarabine and three doses of cyclophosphamide. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of about 30 mg/m ² /day on days -7 through -4 relative to administration of the first portion of a dose of transduced T cells, and further comprises administration of cyclophosphamide at a level of about 600 mg/m ² /day on days -6 through -4 relative to administration of the first portion of a dose of transduced T cells. In some embodiments, the lymphodepleting chemotherapy regimen comprises administration of fludarabine at a level of 30 mg/m ² /day on days -7 through -4 relative to administration of the first portion of a dose of transduced T cells, and further comprises administration of cyclophosphamide at a level of 600 mg/m ² /day on days -6 through -4 relative to administration of the first portion of a dose of transduced T cells.

EXAMPLES

[00399] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1: Dose response in an animal model

[00400] The objective of this study was to evaluate the dose dependent activity of TC-510 (MSLN TRuC/PD-lxCD28) in MHC Class I/II deficient NSG mice bearing large subcutaneously (SC) implanted MSTO-MSLN-PDLl-Luc tumors.

[00401] MHC Class I/II deficient NSG mice were subcutaneously (SC) implanted with MSTO- MSLN-PDLl-Luc cells on Day -28. Once the tumors were established (350-450 mm ³ ), tumor-bearing mice were sorted into treatment cohorts of n=8 mice, and treated with 1.5xl0 ⁶ , l.OxlO ⁶ , 0.5xl0 ⁶ or O. lxlO ⁶ transduced TC-510 T cells, 3xl0 ⁶ non-transduced (NT) control T cells or Vehicle, followed by tumor growth measurement. Mice were monitored for tumor growth during the study and body weight data was collected. Daily clinical observations were performed throughout the study.

[00402] Mice receiving TC-510 cells at the highest doses of 1.5xl0 ⁶ and l.OxlO ⁶ had dramatic tumor regression beginning on day 10 with 7/8 mice tumor-free by day 24 and 8/8 mouse tumor-free by Day 28. Mice receiving the mid-dose of 0.5xl0 ⁶ TC-510 had complete tumor regression of all mice (8/8) by day 28. In contrast, mice treated with the lowest dose of 0. IxlO ⁶ TC-510 T cells had only a slowing of tumor growth with a mean tumor volume of 1100 mm ³ by day 28. The NT control and Vehicle groups had progressive tumor growth with mean tumor volumes of 1900 mm ³ and 2200 mm ³ , respectively, by day 28. The results are provided in FIG. 2A and FIG. 2B. [00403] Body weights were collected twice per week during the study. Mice treated with TC-510 had a higher % change in body weight throughout the study (FIG. 3A-3C). By Day 28, mice treated with 1.5xl0 ⁶ , l.OxlO ⁶ , 0.5xl0 ⁶ and O.lxlO ⁶ TC-510 had a 27%, 25%, 28%, and 26% increase in body weight, respectively. In contrast, mice treated with Vehicle or NT had a 15% or 19% increase in body weight by Day 28. The greater increase in body weight for TC-510 may reflect the fact that mice at the 3 highest doses of TC-510 had complete tumor regression and mice at the lowest dose had some slowing of tumor growth.

[00404] In summary, TC-510 T cell treatment resulted in dose-dependent, anti -turn or activity against large, established SC MSTO-MSLN tumors overexpressing PD-L1. Doses of TC-510 at 1.5xl0 ⁶ and l.OxlO ⁶ transduced T cells/mouse showed strong anti-tumor activity with nearly complete tumor regression by day 24. Mice that received the mid-dose of 0.5xl0 ⁶ transduced T cells/mouse had complete tumor regression by day 28, while the lowest dose of O.lxlO ⁶ transduced T cells resulted in only a slowing of tumor growth. Mice treated with TC-510 had a higher increase in body weight throughout the study compared to mice treated with Vehicle or NT. Based on these results, the minimum anticipated biological effect level (MABEL) of TC-510 in this model was 0.5xl0 ⁶ transduced T cells which is equivalent to 2xl0 ⁶ transduced T cells/kg or a human equivalent dose (HED) of 7.4xl0 ⁷ transduced T cells/m ² .

Example 2: Phase l/II Clinical Trial of TC-510

[00405] This example provides details of a phase 1/2 open-label study to evaluate the safety and efficacy of autologous genetically engineered TC-510 T cells in patients with MSLN-expressing cancers.

[00406] TC-510 T cell is an autologous engineered T cell therapy that uses a patient’s own leukocytes collected via leukapheresis as starting material. The leukocytes are then frozen and shipped to the processing facility. After thawing, non-T cell subpopulations are removed to generate a highly pure T cell population, which then undergoes ex vivo expansion and transduction with a lentiviral vector that encodes for the anti-MSLN TRuC and PD-1/CD28 transgenes. The transduced T cells are subsequently further expanded and then washed and cryopreserved and shipped to the patient’s treating center for administration.

Objectives

[00407] Phase 1 Objectives

Primary

• To evaluate the safety of autologous genetically modified TC-510 T cells in patients with MSLN-expressing metastatic or unresectable solid tumors; and to establish the

- I l l - recommended phase 2 dose (RP2D) according to dose-limiting toxicity (DLT) of defined adverse events (AEs).

Secondary

• To determine the overall response rate (ORR) (complete response [CR] + partial response [PR]) according to Response Evaluation Criteria in Solid Tumors (RECIST) v 1.1 and duration of response (DoR) when TC-510 T cells are administered.

• To determine the disease control rate (DCR), defined as a composite of ORR and stable disease (SD) lasting at least 8 weeks.

• To evaluate the survival benefit of autologous genetically modified TC-510 as assessed by PFS and OS.

[00408] Phase 2 Objectives

Primary

• To evaluate the efficacy of TC-510 in patients with MSLN-expressing metastatic or unresectable solid tumors.

Endpoints: ORR (CR + PR) according to RECIST v 1.1.

DCR (ORR + SD > 812 weeks) according to RECIST v 1.1.

Secondary

• To evaluate the efficacy of autologous genetically modified TC-510 T cells in patients with MSLN-expressing unresectable, metastatic, or recurrent cancers as assessed by TTR, DoR, PFS, and OS.

• To further evaluate the safety of TC-510.

• To explore the clinical benefit of TC-510 re-infusion in patients experiencing SD as best response or in those relapsing after having achieved an initial response to TC-510.

[00409] Exploratory Objectives (Phase 1 and 2)

• To evaluate TC-510 expansion, persistence, and cytokine production in vivo.

• To evaluate key TC-510 product attributes pre- and post-infusion (e.g., phenotype, functionality).

• To evaluate TC-510 infiltration in tumor tissue.

• To evaluate the correlation of metabolic activity of target and non-target lesions post TC-510 infusion and clinical activity.

• To evaluate immune markers in the tumor microenvironment (e.g., PD-L1 expression) before and after TC-510 infusion and their correlation with toxicity and response. To evaluate the changes in health-related quality of life following treatment with TC-510 (in Phase 2).

Study Design

[00410] This first-in-human clinical trial is a phase 1/2 open-label study to evaluate the safety and efficacy of autologous genetically engineered TC-510 T cells in patients with advanced MSLN-expressing cancers. Patients will be screened for general health, performance status, diagnosis, disease stage, and MSLN expression. Following pre-screening, patients meeting all leukapheresis eligibility criteria will undergo a large-volume leukapheresis at the enrolling institution to obtain cells for the manufacture of autologous TC-510 T cells. Patients’ peripheral blood mononuclear cells will be collected and processed at the site and frozen leukocytes will be shipped centrally for further processing. Then, TC-510 transduced T cells will be formulated, cryopreserved, and shipped back to the enrolling institution for infusion.

[00411] The phase 1 portion of the study will evaluate varying doses of TC-510 preceded by lymphodepleting chemotherapy. The lymphodepleting chemotherapy regimen will consist of fludarabine for 4 days (day -7 to day -4) and cyclophosphamide for 3 days (day -6 through day -4).

[00412] It is estimated that 20 to 25 patients will be treated during the dose-escalation phase. A standard 3 + 3 dose escalation strategy will be used to identify the RP2D.

[00413] For the phase 1 portion of the study, TC-510 may be administered via a fractionated regimen at any point in the study if deemed appropriate for safety or efficacy reasons. The TC-510 dose would be fractionated such that one-third (approximately 33%) of the TC-510 dose will be administered on day 0 and, if well tolerated, the remaining two-thirds (approximately 67%) of the dose will be administered on day 7. In the event the initial one-third (33%) dose elicits > grade 3 CRS and/or > grade 2 neurotoxicity, the infusion of the second dose should be delayed until the CRS and/or neurotoxicity regresses to grade 1, or otherwise until treatment is deemed safe by both the treating physician and the Sponsor’s Medical Monitor. A delay of the second infusion by more than 7 days must be approved by the Sponsor. In the event of significant toxicity observed after the first infusion of TC-510, the Sponsor’s Medical Monitor and the Investigator, may determine that the cell dose should be fractionated such that one-third (approximately 33%) of the dose will be administered on day 0 and, if well tolerated, a second-third (approximately 33%) will be administered 7 days later, and if that fraction is well tolerated, the third and final fraction (approximately 33%) will be administered 7 days after the second fraction was received. The same delay or hold rules as described above will apply to this fractionation method. In the phase 2 portion of the study, TC-510 will be administered at the RP2D. [00414] Neutropenia is a common effect of lymphodepleting chemotherapy. The administration of prophylactic granulocyte colony stimulating factor (G-CSF) (e.g., filgrastim) is recommended (but not mandatory) in all patients for management of neutropenia according to published guidelines (e.g., American Society of Clinical Oncology guidelines). G-CSF may be started 24 hours after the administration of lymphodepleting chemotherapy and continued until neutrophil recovery according to institutional practice or physician’s preference. Long-acting (pegylated) G-CSF may be used as per institutional standard practice or physician’s preference. Pegylated GCSF should be given as 1 dose 24 hours after the final dose of cyclophosphamide. Granulocyte-macrophage colony stimulating factor (GM-CSF) is not allowed to be used while on study.

[00415] Both the lymphodepleting chemotherapy as well as the TC-510 infusion may either be given as an outpatient treatment or patients may be hospitalized at the discretion of the Investigator. For the phase 1 portion of the study, all patients should be observed overnight following administration of TC- 510. For the Phase 2 portion of the study, patients may be observed overnight following T cell infusion. [00416] The study will have 2 well-differentiated phases. A schematic summary is provided in FIG. 4. Independent of the phase of the study (phase 1 or phase 2), each patient will follow the same study treatment schedule and procedural requirements. Each patient will proceed through the following study periods:

• Pre-screening/Leukapheresis

• Baseline assessment (Treatment Eligibility) Lymphodepleting chemotherapy

• TC-510 infusion

• Post-infusion follow-up.

[00417] Phase 1

[00418] Patients receiving TC-510 in this dose escalation phase must have one of the following cancer diagnoses: MPM, Serous Ovarian Adenocarcinoma, Pancreatic Adenocarcinoma, Colorectal Cancer, or triple negative breast cancer (TNBC).

[00419] This dose escalation phase will proceed with varying cell doses to determine the RP2D: (50 x 10 ⁶ , 100 x 10 ⁶ , 130 x 10 ⁶ , 160 x 10 ⁶ , and 200 x 10 ⁶ ). All doses mentioned throughout the protocol denote transduced TC-510. A variation on the target dose of 15% (i.e., ± 15%) will be allowed at each dose level.

[00420] Each patient enrolled to the dose-escalation phase of the study will receive TC-510 as either a single infusion or in a fractionated regimen. [00421] Prior to escalation to the next dose level, the SRT, comprised of the Study Sponsor Medical Monitor and at least 2 Study Investigators, will review the safety data and make recommendations on further study conduct of phase 1.

[00422] In the event the RP2D is determined for 1 or more specific indications, dose escalation may continue to determine the indication-specific RP2D for other indications where TC-510 may be tolerated at higher doses. Once the RP2D for a specific indication is determined, enrollment of the Phase 2 specific arm for such indication may commence. Protocol stagger, safety observation and dose escalation rules will continue to apply to patients within the indication-specific dose escalation cohort(s) until the indication-specific RP2D is determined.

[00423] Phase 2

[00424] This phase will evaluate the preliminary antitumor activity (efficacy) and better characterize safety of TC-510 at the selected RP2D. Patients will receive TC-510 at the RP2D and will be enrolled according to their cancer diagnosis to 5 distinct cohorts of 20 patients: MPM, Serous Ovarian Adenocarcinoma, Pancreatic Adenocarcinoma, Colorectal Cancer, and Triple Negative Breast Cancer. Overall, the phase 2 portion of the study will treat 100 patients.

TC-510 T cell Dose and Treatment Schedule

[00425] Phase 1 portion of the study: Each patient will receive a single dose of TC-510. At each dose, TC-510 will be given to at least 3 patients following lymphodepleting chemotherapy (fludarabine 30 mg/m ² /d on days -7 through -4 and cyclophosphamide 600 mg/m ² /d on days -6 through -4). Patients will be enrolled at the dose levels (DL) shown in Table 2 to determine the RP2D:

Table 2. Dose Escalation

[00426] A variation on the target dose of 15% (i.e., ± 15%) will be allowed at each dose level.

[00427] In the event of excessive toxicity at the initial dose level (DL0), the study will resume at DL-

1 (i.e., 25 x io ⁶ transduced T cells on day 0).

[00428] Dose escalation will be directed by the SRT upon review of the safety data.

[00429] Once identified, up to 10 patients may be subsequently treated at the RP2D to further delineate the toxicity profile of TC-510 prior to advancing to the phase 2 portion of the study. [00430] Phase 2 portion of the study: Patients will be stratified according to their cancer diagnosis into 5 cohorts: MPM, serous ovarian cancer adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, and triple negative breast cancer. Within each cohort, patients will receive a single TC-510 infusion at the RP2D determined in the phase 1 portion of the study. Patients experiencing SD as best response (at least 8 weeks post infusion) and those experiencing loss of a response to their initial TC- 510 infusion will be candidates to redosing with TC-510 at the RP2D (or at a lower dose if a new product is not available at the RP2D) following lymphodepleting chemotherapy.

Definition of Dose Limiting Toxicity

[00431] A DLT is defined as any of the following TC-510 T cell-related events with onset within the ensuing 28 days following TC-510 T cell infusion:

• Any Common Terminology Criteria for Adverse Events (CTCAE) grade 5 organ toxicity.

• Any CTCAE grade 4 organ toxicity (cardiac, dermatologic, gastrointestinal, hepatic, pulmonary, renal/genitourinary, or neurologic) with an attribution of definitely or probably related to the TC- 510 T cell infusion that does not improve to grade < 2 within 4 weeks.

• Any grade 3 or greater autoimmune toxicity related to TC-510 T cell therapy.

• Grade 4 CRS that does not improve to < 3 within 72 hours. If grade 4 CRS resolves to grade 3 within 72 hours, then the grade 3 CRS must further resolve to grade < 2 within the following 11 days so that the time between grade 4 CRS and its resolution to grade <2 does not exceed 14 days.

• Grade 4 neurotoxicity that does not improve to < 3 within 72 hours. If grade 4 neurotoxicity resolves to grade 3 within 72 hours, then the grade 3 neurotoxicity must further resolve to grade < 2 within the following 11 days so that the time between grade 4 neurotoxicity and its resolution to grade <2 does not exceed 14 days.

[00432] The following adverse events will NOT be considered DLTs:

• Grade 1 to 4 toxicities expected with lymphodepleting chemotherapy (e.g., cytopenias).

• Fever grade < 3.

• Myelosuppression (includes bleeding in the setting of platelet count less than 50 x 10 ⁹ /L and documented bacterial infections in the setting of neutropenia), defined as lymphopenia, decreased hemoglobin, neutropenia and/or thrombocytopenia.

• Immediate hypersensitivity reactions occurring within 2 hours of TC-510 T cell infusion (related to cell infusion) that are reversible to a grade 2 or less within 24 hours of TC-510 T cell administration with standard therapy.

Leukapheresis Inclusion/Exclusion Criteria [00433] Patients will be assessed for and must meet leukapheresis eligibility criteria for leukapheresis at the Leukapheresis Eligibility visit.

Leukapheresis Inclusion Criteria

[00434] A patient must meet the following inclusion criteria to be eligible to undergo leukapheresis:

• Patient (or legally authorized representative) has voluntarily agreed to participate by giving written informed consent in accordance with International Conference on Harmonisation (ICH) Good Clinical Practice (GCP) guidelines and applicable local regulations.

• Patient is > 18 years of age at the time the Informed Consent is signed.

• Patient has a pathologically confirmed diagnosis of either MPM, Serous Ovarian Adenocarcinoma (patients with Serous Ovarian, Fallopian Tube or Primary Peritoneal cancers will be permitted), Pancreatic Adenocarcinoma, Colorectal Cancer, or TNBC. o For patients with triple negative breast cancer, treatment sites must provide proof of ER-negative and PR-negative status (in both cases defined as < 1%). Her2-negative status is defined as 0, 1+, or 2+ by immunohistochemistry (IHC). If IHC 2+, a negative in situ hybridization (FISH, CISH, or SISH) test may be required by local laboratory testing.

• Patient’s tumor expresses MSLN on 50% of tumor cells with 1+, 2+, and/or 3+ intensity by immunohistochemistry at a central laboratory: o A banked tumor biopsy is allowed at pre-screening if obtained within the prior 12 months, otherwise a fresh tumor biopsy should be obtained.

• Patient has advanced (metastatic or unresectable) cancer. Unresectable refers to a tumor lesion in which clear surgical excision margins cannot be obtained without leading to significant functional compromise.

• Patient has at least 1 lesion that meets evaluable and measurable criteria defined by RECIST v 1.1 after the pre-screening fresh-tissue biopsy has been performed. Patients who have received prior local therapy (including but not limited to embolization, chemoembolization, radiofrequency ablation, or radiation therapy) are eligible provided measurable disease falls outside of the treatment field or is within the field and has shown > 20% growth in size since post-treatment assessment.

• Prior to TC-510 infusion, patients must have received at least 1 but no more than 5 systemic therapies for metastatic or unresectable disease unless otherwise specified below: o MPM: Patients must have received standard frontline therapy (e.g., platinum-based chemotherapy or checkpoint inhibitor therapy). o Pancreatic Adenocarcinoma: Patients must have received frontline therapy with fluorouracil-based (e.g., FOLFIRINOX) or gemcitabine-based (e.g. gemcitabine ± Nab-paclitaxel) regimens for advanced disease or have elected not to pursue standard front-line therapy. o Serous Ovarian Adenocarcinoma: Patients must have received a platinum-based regimen. Or if they are carriers of a BRCA1/2 mutation they must have received at least a PARP inhibitor. o Triple Negative Breast Cancer: Must have received at least 1 prior systemic anti-cancer therapy, including a PARP inhibitor for advanced TNBC with a germline mutation in BRCA1/2, and the combination of atezolizumab with nab-paclitaxel for advanced TNBC that expresses PD-L1 on immune cells within the tumor. o Colorectal Cancer: Must have progressed after at least 1 prior standard systemic anticancer therapy (e.g., oxaliplatin or irinotecan-based regimens ± bevacizumab), including cetuximab or panitumumab in patients with wild type KRAS or immune checkpoint inhibitors in patients with MSI-H or dMMR colorectal cancer.

• Patient has an Eastern Cooperative Oncology Group performance status 0 or 1.

• The patient must not have required a paracentesis or thoracentesis within the preceding

4 weeks nor be projected to require a paracentesis or thoracentesis within the next 8 weeks. Patients with catheters in place for frequent drainage may be allowed. Specific cases are to be discussed with the Medical Monitor and Sponsor.

• No evidence of bowel obstruction within the last 8 weeks.

• Subject is fit for leukapheresis and has adequate venous access for the cell collection.

• FPCP must have a negative urine or serum pregnancy test (FPCP is defined as premenopausal and not surgically sterilized). FPCP must agree to use effective birth control or to abstain from heterosexual activity throughout the study, starting on the day of first dose of lymphodepleting chemotherapy through 12 months post TC-510 infusion or for 4 months after there is no evidence of persistence of gene modified cells in the blood, whichever is longer. Effective contraceptive methods include intra-uterine device, oral or injectable hormonal contraception, or 2 adequate barrier methods (e.g., diaphragm with spermicide, cervical cap with spermicide, or female condom with spermicide). Spermicides alone are not an adequate method of contraception. o Or, Male patients must be surgically sterile or agree to use a double barrier contraception method or abstain from heterosexual activity with a female of childbearing potential starting at the first dose of protocol-defined treatment and for 4 months thereafter or longer (if indicated in the country specific monograph/label for cyclophosphami de) .

• Subject must have adequate organ function as indicated by the laboratory values in Table 3:

Table 3. Laboratory Values Indicating Sufficient Organ Function

Leukapheresis Exclusion Criteria [00435] A patient meeting any of the following exclusion criteria is not eligible to undergo leukapheresis:

• Inability to follow the procedures of the study (e.g., due to language problems, psychological disorders, dementia, confusional state).

• Patient has received or plans to receive the following therapy/treatment prior to leukapheresis:

• Cytotoxic chemotherapy within 3 weeks of leukapheresis

• Corticosteroids: therapeutic doses of steroids must be stopped > 72 hours prior to leukapheresis. Use of inhaled steroids or topical cutaneous steroids is not exclusionary. Corticosteroid therapy at a pharmacologic dose (> 5 mg/day of prednisone or equivalent doses of other corticosteroids) and other immunosuppressive drugs should be avoided until 3 months after TC-510 T cell administration, unless medically indicated to treat new toxicity. Physiological replacement doses of steroids (up to 5 mg/day of prednisone equivalent, or higher if warranted by the patient’s BMI) may be allowed.

• Immunosuppression: any other immunosuppressive medication must be stopped > 4 weeks prior to leukapheresis, including calcineurin inhibitors, methotrexate or other chemotherapy drugs, mycophenolate, steroids (see above), rapamycin, thalidomide, or immunosuppressive antibodies such as rituximab, anti-tumor necrosis factor, anti-interleukin (IL) 6 or anti-IL6R.

• Use of an anti-cancer vaccine within 2 months in the absence of tumor response. The patient should be excluded if their disease is responding to an experimental vaccine given within 6 months;

• Any previous gene therapy using an integrating vector;

• Tyrosine kinase inhibitor (e.g., EGFR inhibitors), ), PARP inhibitors (e.g., olaparib, rucaparib, niraparib), or KRAS G12C inhibitors (e.g., sotorasib, adagrasib) within 72 hours;

• Any previous allogeneic hematopoietic stem cell transplant;

• Investigational treatment or clinical trial within 4 weeks or 5 half-lives of investigational product, whichever is shorter.

• Coronavirus disease 2019 (Covid-19; SARS-CoV-2) vaccine dose within 4 weeks from estimated date of leukapheresis, unless approved by TCR2 Therapeutics Medical Monitor.

• CNS disease/brain metastases:

• Patients with leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases: patients are eligible if they have completed their treatment, have recovered from the acute effects of radiation therapy or surgery prior to study entry, and a) have no evidence of brain metastases post treatment or b) are asymptomatic, have discontinued corticosteroid treatment or anti-seizure medications for these metastases for at least4 weeks and have radiographically stable CNS metastases without associated edema or shift for at least 3 months prior to study entry (note: prophylactic anti-seizure medications are acceptable; up to 5 mg per day of prednisone or equivalent will be allowed, or higher if warranted by the patient’s BMI).

• Patient has any other prior or concurrent malignancy with the following exceptions:

• Adequately treated basal cell or squamous cell carcinoma (adequate wound healing may be required prior to study entry).

• In situ carcinoma of the cervix or breast, treated curatively and without evidence of recurrence for at least 12 months prior to the study.

• Treated non-melanoma skin cancer.

• Stage 0 or 1 melanoma completely resected at least 12 months prior to the study.

• Successfully treated organ-confined prostate cancer with no evidence of progressive disease based on PSA levels and are not on active therapy.

• A primary malignancy which has been completely resected and in complete remission for 5 years.

• Other malignancies deemed unlikely to be of clinical significance during TC-510 therapy by the Principal Investigator and as approved by the Sponsor.

• Patient has active infection with HIV, hepatitis B virus, HCV, or HTLV as defined below:

• Positive serology for HIV, HTLV-1, or HTLV-2.

• Active hepatitis B infection as demonstrated by test for hepatitis B surface antigen. Patients who are hepatitis B surface antigen negative but are hepatitis B core antibody positive must have undetectable hepatitis B DNA and receive prophylaxis against viral reactivation.

• Active hepatitis C infection as demonstrated by hepatitis C RNA test. Patients who are HCV antibody positive will be screened for HCV RNA by any reverse transcription PCR or branched DNA assay. If HCV antibody is positive, eligibility will be determined based on a negative screening RNA value.

• Patient with pulse oximetry < 90% on room air or has one of the following will be excluded

• Patients with radiographic evidence of underlying interstitial lung disease. Patients with active interstitial lung disease/pneumonitis or a history of interstitial lung disease/pneumonitis requiring therapy with systemic corticosteroids.

• History of autoimmune or immune mediated disease such as multiple sclerosis, lupus, rheumatoid arthritis, inflammatory bowel disease, Hashimoto’s thyroiditis, or small vessel vasculitis. Treatment Inclusion/Exclusion Criteria

[00436] A patient must meet the following treatment inclusion criteria to be eligible to proceed with first protocol defined therapy (e.g. Lymphodepletion). Treatment Eligibility will be formally assessed at Baseline. For retreatment with TC-510, the following criteria also apply.

Treatment Inclusion Criteria

[00437] A patient must meet the following inclusion criteria to be eligible to receive therapy on this study:

• Patient (or legally authorized representative) has voluntarily agreed to participate by giving written informed consent in accordance with International Conference on Harmonisation Good Clinical Practice guidelines and applicable local regulations.

• Patient has agreed to abide by all protocol required procedures including study -related assessments, and management by the treating institution for the duration of the study and LTFU.

• Patient is > 18 years of age at the time the Informed Consent is signed.

• Patient has a pathologically confirmed diagnosis of either MPM, Serous Ovarian Adenocarcinoma (patients with Serous Ovarian, Fallopian Tube or Primary Peritoneal cancers will be permitted), Pancreatic Adenocarcinoma, Colorectal Cancer, or TNBC.

• For patients with TNBC, treatment sites must provide proof of ER-negative and PR-negative status (in both cases defined as < 1%). Her2-negative status is defined as 0, 1+, or 2+ by IHC. If IHC 2+, a negative in situ hybridization (FISH, CISH, or SISH) test may be required by local laboratory testing.

• Patient’s tumor expresses MSLN on > 50% of tumor cells with 1+, 2+, and/or 3+ intensity by immunohistochemistry at a designated central laboratory:

• If the patient had a fresh biopsy during pre-screening, that biopsy sample may be used for Baseline MSLN testing. If archival tissue was used during pre-screening, the patient must undergo a fresh biopsy, unless deemed unsafe by the PI and/or the lesion is inaccessible and upon discussion with the medical monitor. For TC-510 retreatment, fresh tissue acquired within 2 months of retreatment of the first protocol defined therapy must be tested for MSLN expression.

• Patient has advanced (metastatic or unresectable) cancer. Unresectable refers to a tumor lesion in which clear surgical excision margins cannot be obtained without leading to significant functional compromise.

• Patient has at least 1 lesion that meets evaluable and measurable criteria confirmed RECIST v 1.1 after the pre-TC-510 infusion fresh-tissue biopsy has been performed. Patients who have received prior local therapy (including but not limited to embolization, chemoembolization, radiofrequency ablation, or radiation therapy) are eligible provided measurable disease falls outside of the treatment field or within the field and has shown >20% growth in size since post-treatment assessment.

• The patient must not have required a paracentesis or thoracentesis within the preceding 4 weeks of TC-510 infusion nor be projected to require a paracentesis or thoracentesis within the next 8 weeks. Patients with catheters in place for frequent drainage may be allowed.

• Prior to TC-510 infusion, patients must have received at least 1 but no more than 5 systemic therapies for metastatic or unresectable disease unless where specified below:

• MPM: Patients must have received standard frontline therapy (e.g., platinum-based chemotherapy or checkpoint inhibitor therapy).

• Pancreatic Adenocarcinoma: Patients must have received frontline therapy with fluorouracilbased (e.g., FOLFIRINOX) or gemcitabine-based (e.g. gemcitabine ± Nab-paclitaxel) regimens for advanced disease or have elected not to pursue standard front-line therapy.

• Serous Ovarian Adenocarcinoma: Patients must have received a platinum-based regimen. Or if they are carriers of a BRCA1/2 mutation they must have received at least a PARP inhibitor.

• Triple Negative Breast Cancer: Must have received at least 1 prior systemic anti-cancer therapy, including a PARP inhibitor for advanced TNBC with a germline mutation in BRCA1/2, and the combination of atezolizumab with nab-paclitaxel for advanced TNBC that expresses PD-L1 on immune cells within the tumor.

• Colorectal Cancer: Must have progressed after at least 1 prior standard systemic anti-cancer therapy (e.g., oxaliplatin or irinotecan-based regimens ± bevacizumab), including cetuximab or panitumumab in patients with wild type KRAS or immune checkpoint inhibitors in patients with MSI-H or dMMR colorectal cancer.

• Patient has an Eastern Cooperative Oncology Group performance status 0 or 1.

• All patients must have undergone a rapid influenza diagnostic test and/or a respiratory viral panel (including coronavirus disease 2019 (Covid-19; SARS-CoV-2) as per Institutional guidelines within 14 days prior to the first protocol defined therapy. If the patient is positive for influenza, oseltamivir phosphate or zanamivir should be administered for 10 days (see Tamiflu® or Relenza® package insert for dosing). The patient must complete their 10-day treatment course prior to receiving TC-510.

• For patients residing in the US, Canada, Europe and Japan, influenza testing may be required during the months of October through May (inclusive). • For patients residing in the southern hemisphere such as Australia, influenza testing may be required during the months of April through November (inclusive).

• For patients with significant international travel, both calendar intervals above may need to be considered.

• In the event a patient tests positive for coronavirus disease 2019 (Covid- 19; SARS-CoV-2), TC-510 infusion should be delayed until the patient is asymptomatic and deemed fit for infusion by the treating physician.

• Patient has a left ventricular ejection fraction > 45% as measured by resting echocardiogram, with no clinically significant pericardial effusion.

• FPCP must have a negative urine or serum pregnancy test (FPCP is defined as premenopausal and not surgically sterilized). FPCP must agree to use effective birth control or to abstain from heterosexual activity throughout the study, starting on the day of first dose of lymphodepl eting chemotherapy through 12 months post TC-510 infusion. Effective contraceptive methods include intra-uterine device, oral or injectable hormonal contraception, or 2 adequate barrier methods (e.g., diaphragm with spermicide, cervical cap with spermicide, or female condom with spermicide). Spermicides alone are not an adequate method of contraception.

• Or: Male patients must be surgically sterile or agree to use a double barrier contraception method or abstain from heterosexual activity with a female of childbearing potential starting at the first dose of protocol -defined treatment and for 4 months thereafter or longer (if indicated in the country specific monograph/label for cyclophosphamide).

• Patient must have adequate organ function as indicated by the laboratory values in Table 3 above. Treatment Exclusion Criteria

[00438] A patient meeting any of the following exclusion criteria is not eligible for participation in the treatment portion of this study:

• Inability to follow the procedures of the study (e.g., due to language problems, psychological disorders, dementia, confusional state).

• Known or suspected non-compliance, drug, or alcohol abuse.

• Participation in another study with investigational drug within the 28 days or 5 half-lives of the drug, whichever is shorter, preceding and during the present study.

• Patient is pregnant (or intends to become pregnant during the course of the study) or breastfeeding.

• Patient has received or plans to receive the following therapy/treatment prior to the first protocol- defined therapy:

• Cytotoxic chemotherapy within 3 weeks of TC-510 T cell infusion. • Corticosteroids: therapeutic doses of steroids must be stopped at least 2 weeks prior to TC-510 T cell infusion. Use of inhaled steroids or topical cutaneous steroids is not exclusionary. Corticosteroid therapy at a pharmacologic dose (> 5 mg/day of prednisone or equivalent doses of other corticosteroids) and other immunosuppressive drugs should be avoided until 3 months after TC-510 T cell administration, unless medically indicated to treat new toxicity. Physiological replacement doses of steroids (up to 5 mg/day of prednisone equivalent, or higher if warranted by the patient’s BMI) may be allowed.

• Any other immunosuppressive medication must be stopped > 4 weeks prior to first protocol defined treatment, including (but not limited to) calcineurin inhibitors, methotrexate or other chemotherapy drugs, mycophenolate, steroids (see above), rapamycin, thalidomide, or immunosuppressive antibodies such as rituximab, anti-tumor necrosis factor, anti-interleukin (IL) 6 or anti-IL6R.

• Anti-PD-1 monoclonal antibody therapy must be stopped > 4 weeks prior to first protocol- defined treatment.

• Use of an anti-cancer vaccine within 2 months in the absence of tumor response. The patient should be excluded if their disease is responding to an experimental vaccine given within 6 months;

• Any previous gene therapy using an integrating vector;

• Small molecule tyrosine kinase inhibitors (e.g., EGFR inhibitors), PARP inhibitors (e.g., olaparib, rucaparib, niraparib), or KRAS G12C inhibitors (e.g., sotorasib, adagrasib) within 72 hours;

• Any previous allogeneic hematopoietic stem cell transplant;

• Investigational treatment or clinical trial within 4 weeks or 5 half-lives of investigational product, whichever is shorter;

• Radiotherapy to the target lesions within 3 months prior to lymphodepleting chemotherapy. A lesion with unequivocal progression may be considered a target lesion regardless of time from last radiotherapy dose. NOTE: There is no washout period for palliative radiation to non-target lesions;

• Hepatic radiation, chemoembolization, and/or radiofrequency ablation within 4 weeks.

• Current anticoagulative therapy (excluding deep vein thrombosis prophylaxis).

• Immune therapy (e.g. monoclonal antibody therapy, checkpoint inhibitors) within 4 weeks. • Toxicity from previous anti-cancer therapy that has not recovered to < grade 1 (except for nonclinically significant toxicities, e.g., alopecia, vitiligo). Patients with grade 2 toxicities that are deemed stable or irreversible (e.g., peripheral neuropathy) can be enrolled.

• History of allergic reactions attributed to compounds of similar chemical or biologic composition to fludarabine, cyclophosphamide, or other agents used in the study.

• History of autoimmune or immune mediated disease such as multiple sclerosis, lupus, rheumatoid arthritis, inflammatory bowel disease, Hashimoto’s thyroiditis, or small vessel vasculitis.

• Maj or surgery (other than diagnostic surgery) within 4 weeks prior to first protocol defined therapy, minor surgery including diagnostic surgery within 2 weeks (14 days) excluding central intravenous port placements and needle aspirate/core biopsies. Radio frequency ablation or transcatheter arterial chemoembolization within 6 weeks prior to enrollment.

• Central nervous system (CNS) disease/brain metastases:

• Patients with leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases: patients are eligible if they have completed their treatment, have recovered from the acute effects of radiation therapy or surgery prior to study entry, and a) have no evidence of brain metastases post treatment or b) are asymptomatic, have discontinued corticosteroid treatment or anti-seizure medications for these metastases for at least 4 weeks and have radiographically stable CNS metastases without associated edema or shift for at least 3 months prior to study entry (note: prophylactic anti-seizure medications are acceptable; up to 5 mg/day of prednisone or equivalent will be allowed; higher doses may be allowed if warranted due to patient BMI).

• Patient has any other prior or concurrent malignancy with the following exceptions:

• Adequately treated basal cell or squamous cell carcinoma (adequate wound healing may be required prior to study entry)

• In situ carcinoma of the cervix or breast, treated curatively and without evidence of recurrence for at least 12 months prior to the study

• Treated non-melanoma skin cancer

• Stage 0 or 1 melanoma completely resected at least 12 months prior to enrollment

• Successfully treated organ-confined prostate cancer with no evidence of progressive disease based on prostate specific antigen (PSA) levels and are not on active therapy

• A primary malignancy which has been completely resected and in complete remission for > 5 years • Malignancies deemed unlikely to be of clinical significance during TC-510 T cell therapy by principal investigator and as approved by sponsor

• Patient has an electrocardiogram showing a clinically significant abnormality at screening or showing an average QTc interval > 450 msec in males and > 470 msec in females (> 480 msec for patients with bundle branch block). Either Fridericia’s or Bazett’s formula may be used to correct the QT interval.

• Patient has uncontrolled intercurrent illness including, but not limited to:

• Clinically significant cardiac disease defined by congestive heart failure New York Heart Association class 3 or class 4;

• Uncontrolled clinically significant arrhythmia;

• Acute coronary syndrome (angina or myocardial infarction), stroke, or peripheral vascular event in the last 6 months;

• Interstitial lung disease (patients with existing pneumonitis as a result of radiation are not excluded; however, patients must not be oxygen dependent as demonstrated by oxygen saturation < 90% on room air);

• Liver cirrhosis.

• Patient has active infection with human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus (HCV), or human T-lymphotropic virus (HTLV) as defined below:

• Positive serology for HIV, HTLV- 1 , or HTL V-2;

• Active hepatitis B infection as demonstrated by test for hepatitis B surface antigen. Patients who are hepatitis B surface antigen negative but are hepatitis B core antibody positive must have undetectable hepatitis B deoxyribonucleic acid (DNA) and receive prophylaxis against viral reactivation;

• Active hepatitis C infection as demonstrated by hepatitis C ribonucleic acid (RNA) test. Patients who are HCV antibody positive will be screened for HCV RNA by any reverse transcription polymerase chain reaction (PCR) or branched DNA assay. If HCV antibody is positive, eligibility will be determined based on a negative screening RNA value.

Study Treatment

[00439] TC-510 is an engineered autologous ACT. Patients who complete screening procedures and who meet leukapheresis eligibility criteria as defined above will be eligible to undergo leukapheresis to obtain starting material for the manufacture. A large-volume non-mobilized PBMC collection will be performed (12- to 15-liter apheresis) according to institutional standard procedures for collection of the starting material. The goal will be to collect approximately 5 to 10 * 10 ⁹ total PBMCs (minimum collection goal 1.5 x io ⁷ PMBC/kg). The leukapheresed cells will then be frozen and transported either the same day or overnight to the TCR2 Therapeutics approved Cell Processing Facility (CPF). In cases where the minimum number of PBMCs are not collected or the manufacturing of sufficient TC-510 is not successful, a second leukapheresis may be performed. Citrate anticoagulant should be used during the procedure and prophylaxis against the adverse effects of this anticoagulant (e.g., CaC12 infusions) may be employed at the Investigator’s discretion.

[00440] Upon arrival at the CPF, each patient’s leukapheresed product will be processed to enrich for the T cells containing PBMC fraction. T cells will be then stimulated to expand and transduced with a lentiviral vector to introduce the TC-510 transgene to obtain TC-510. Transduced T cells (i.e., TC- 510) are then expanded and cryopreserved to generate the investigational product per CPF Standard Operating Procedures (SOPs). Once the TC-510 product has passed release tests, the CPF will ship the product back to the treating facility.

[00441] Patients must meet treatment eligibility at the Baseline visit (Visit 4). TC-510 will be administered first during the phase 1 portion of the study (i.e., dose-escalation phase) at the initial dose of 50 x io ⁶ transduced T cells (i.e., DL0). The dose-escalation phase will evaluate varying TC-510 cell doses. During the phase 2 portion of the study, patients will receive TC-510 at the RP2D. A dose range of ± 15% of the target dose may be administered.

[00442] TC-510 will be supplied cryopreserved in cryostorage bags. The product in the bag will be opaque, with cream to white color. The cryostorage bags containing TC-510 will arrive frozen in a liquid nitrogen dry shipper. The bags must be stored in vapor phase of liquid nitrogen and the product remains frozen until the patient is ready for treatment to assure viable live autologous cells are administered to the patient. Several inactive ingredients are added to the product to assure viability and stability of the live cells through the freezing, thawing, and infusion process. Each bag contains a patient-specific product, and the intended patient will be identified by patient ID number. The product should be thawed and administered to the patient as specified in the investigational product manual. The product must not be thawed until the patient is ready for the infusion. In case of accidental overdose, treatment should be supportive. Corticosteroid therapy and/or tocilizumab may be considered if any dose is associated with severe toxicity.

[00443] Bridging Therapy: During the time between leukapheresis and starting protocol-defined therapy, patients may receive therapy aimed at maintaining control over or stabilizing his or her disease. The therapy used during this time will be considered a “bridging” therapy aimed at bridging the gap between prior treatment and treatment with TC-510. The protocol will allow for patients to receive appropriate therapies for the indication and disease status. The patients must be off-treatment for specific timeframes based on the product mechanism as described in the eligibility criteria. [00444] Lymphodepleting Chemotherapy: Lymphodepleting chemotherapy will be supplied by the investigative site unless otherwise noted. Sites should refer to the current product label for guidance on packaging, storage, preparation, administration, and toxicity management associated with the administration of both agents.

[00445] Clinical sites are allowed to follow institutional guidelines for administration, hydration, and monitoring parameters pertaining to the chemotherapy agents described in this protocol. In the absence of institutional standards, suggested guidelines are detailed below.

[00446] Patients will receive a non-myeloablative chemotherapy regimen consisting of fludarabine 30 mg/m ² /day on days -7 through -4 (i.e., 4 doses) and cyclophosphamide 600 mg/m ² /day on days -6 through -4 (i.e., 3 doses) (Table 4). In the event the patient has an unforeseeable delay or missed dosing day for this lymphodepleting regimen, the missed dose will be administered, and the TC-510 dosing day will be moved back, maintaining the last dosing day at day -4 relative to TC-510 infusion. The intent of this regimen is to induce lymphocyte depletion to promote an optimal environment for the expansion of TC-510 in vivo.

Table 4. Lymphocyte depletion treatment schedule

[00447] Fludarabine: Fludarabine phosphate is a synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite. The dose of fludarabine will be adjusted for patients with renal dysfunction as shown in Table 5. For patients >65 years of age, the measured creatinine clearance value at baseline must be used to adjust the fludarabine dose.

[00448] Cyclophosphamide and Mesna: Cyclophosphamide is a nitrogen mustard-derivative alkylating agent. Following conversion to active metabolites in the liver, cyclophosphamide functions as an alkylating agent; the drug also possesses potent immunosuppressive activity. The serum half-life after IV administration ranges from 3 to 12 hours; the drug and/or its metabolites can be detected in the serum for up to 72 hours after administration. Mesna will be given to cover the duration of cyclophosphamide chemotherapy according to institutional guidelines.

[00449] TC-510 Infusion: On day 0 of the study, patients participating in the phase 1 portion of the study will receive TC-510 within the dose range of 50 x 10 ⁶ to 200 x 10 ⁶ transduced cells by IV infusion.

[00450] The recommended dose for patients participating in the phase 2 portion will be determined at the end of the dose-escalating phase 1. [00451] TC-510 is a patient-specific product. Upon receipt, verification that the product and patientspecific labels match the intended patient information is essential. Do not infuse the product if the information on the patient-specific label does not match the intended patient. Prior to infusion, 2 clinical personnel in the presence of the patient will independently verify and confirm that the information on the infusion bag label is correctly matched to the patient, in accordance with institutional practice for the administration of cell products. Thirty to 60 minutes prior to cell infusion, patients will be premedicated against potential infusion reactions with antihistamines and acetaminophen (paracetamol) as per institutional practice. Steroids must not be administered as premedication for T cell infusion due to their lymphotoxic potential against the TC-510 product.

[00452] TC-510 must not be thawed until immediately prior to infusion. The product can be thawed either in a water bath at the patient’s bedside or with a device such as GE ViaThaw in a centralized facility, according to institutional standard procedures. The cells must be infused without delay and, if thawed centrally, must be transported to the patient by appropriately trained clinical staff, to preserve the chain of custody. The product must not be washed or otherwise processed.

[00453] It is expected that the infusion will commence within approximately 10 minutes of thawing (or within 10 minutes of receipt if thawed centrally) and complete within 45 minutes of thawing (or receipt from centralized thawing facility) to minimize exposure of the TC-510 product to cryoprotectant. If the cells are provided in multiple bags, the additional bag(s) must not be thawed until half of the first bag has been infused without reaction.

[00454] TC-510 is to be administered using a dual spike infusion set by gravity over 15 to 30 minutes (in the absence of reaction) via non-filtered tubing. The bag should be gently agitated during infusion to avoid cell clumping. Infusion pumps must not be used. For administration of TC-510, 100 to 250 mL of 0.9% NaCl should be connected to the second lumen of the infusion set, used to prime the line, and then the lumen should be closed. On completion of the infusion of a cell bag, the main line should be closed and approximately 50 mL NaCl should be transferred into the cell bag and then infused to minimize cell loss. This process should be repeated for each bag if multiple bags are provided. On completion of the cell infusion, the set should be flushed using additional saline from the attached bag. In the event institutional practice requires a single spike infusion set, the line must be flushed with 0.9% NaCl once the infusion is complete. In the event of an adverse reaction to TC-510 infusion, the infusion rate should be reduced, and the reaction managed according to institutional standard procedures. Corticosteroids should be avoided unless medically required. In the event a patient develops a febrile episode following the infusion, appropriate cultures and medical management should be initiated, with attention to the initiation of empirical antibiotic treatment in the case of febrile neutropenia. The infusion of the TC-510 product may be delayed by up to 5 days should a patient develop significant chemotherapy-related complications, if it is judged to be in the patient’s best interest by the Investigator. Cytopenias alone should not be a reason to delay TC-510 infusion unless complications (e.g., uncontrolled infection or bleeding) are present. The volume of TC-510 product infused, the thaw time, the start time, and the stop time will all be noted in the patient medical record. Vital signs will be recorded within 10 minutes prior to the infusion and at 5 (± 3 minutes), 15 (± 3 minutes), and 30 minutes (± 5 minutes) and at 1 (± 5 minutes), 1.5 (± 5 minutes), 2 (± 5 minutes), and 4 hours (± 5 minutes) after the infusion has started. For the phase 1 portion of the study, patients should be observed overnight following the infusion of TC-510.

[00455] The same infusion process will be followed for retreatment with TC-510 (phase 2 only).

Criteria for Evaluation: Safety

[00456] This will be the first-in-human trial of TC-510 and therefore, no clinical safety information with this therapeutic is currently available. However, TC-510 represents the third TRuC T cell product to be studied in subjects with cancer, the first 2 are gavo-cel and TC-110. TC-510 results from engineering gavo-cel T cells to have them express a PD-1 :CD28 switch. A series of common toxicities have been identified across a growing number of trials involving autologous T cells genetically engineered to express either chimeric antigen receptors or T cell receptors targeting tumor antigens. The most important of such toxicities are CRS, neurotoxicity, graft-versus-host disease (GvHD), and myelosuppression. CRS events have also been reported in patients receiving gavo-cel therapy but they have been generally manageable upon institution of standard CRS management measures (e.g., corticosteroids, tocilizumab). No GVHD events and only 1 low grade self-limited neurotoxic event has been reported among patients treated with gavo-cel to date. To manage the risk of CRS and other potential TC-510 mediated toxicities, specific adverse event (AE) pages in the electronic case report form (eCRF) have been implemented to carefully document the events and to enable evaluation and identification of potential risk factors. To help Investigators manage TC-510 mediated toxicities, treatment guidelines based on published literature which are included in the protocol have been developed. Standard protocol guidance on the management of myelosuppression, including a recommendation to consult with physicians with expertise in bone marrow transplant, infectious diseases, and the management of aplastic anemia, has also been developed.

[00457] The theoretical risks of replication competent lentivirus (RCL) and insertional oncogenesis will be monitored in accordance with FDA and European Medicines Agency (EMEA) guidance in the long-term follow-up study protocol.

[00458] Statistical Methods: The co-primary clinical endpoints for efficacy are ORR, defined as the proportion of patients with a CR or PR via independently reviewed RECIST v 1.1 relative to the total number of patients in the analysis population, and DCR, defined as the ORR plus the proportion of patients with SD for at least 8 weeks via independently reviewed RECIST v 1.1 relative to the total number of patients in the analysis population.

[00459] Study Populations

• Intent-to-treat population: all patients who have signed the informed consent form and have undergone leukapheresis.

• Modified intent-to-treat (mITT) population: all patients enrolled and treated with the planned dose of study drug.

• DLT Evaluable population: (phase 1 only) patients treated in the phase 1 portion of the study who received study drug and were followed for at least 28 days post-administration or who have a DLT within 28 days of the first study drug dose.

• Safety Set: all patients treated with any dose of study drug.

[00460] The primary analysis population for safety and efficacy will be the mITT defined as all patients who received a TC-510 T cell infusion.

[00461] No specific statistical hypotheses are being evaluated with respect to the primary, secondary, or exploratory objectives and endpoints. The primary focus will be on determining the safety profile and a preliminary efficacy profile of TC-510 T cells, as well as on establishing the kinetics of TC-510 T cells following infusion. All analyses will be descriptive and exploratory. Descriptive statistics on continuous data will include means, medians, standard deviations, and ranges. Categorical data will be summarized using frequency counts and percentages. Graphical summaries of the data may be presented. Assessments will be displayed by cohort and time as well as across cohorts for specific parameters. Time to event endpoints will be summarized and displayed graphically using Kaplan- Meier (KM) methodology to estimate the median, and the 25 ^th and 75 ^th percentiles. Two-sided 80% confidence intervals will be produced. Overall survival may be assessed at fixed time points such as 1 year and 2 years using KM methods.

[00462] Study Treatment and Concomitant Therapy: During the course of the study, investigators may prescribe any concomitant medications or treatment deemed necessary to provide adequate supportive care except those medications listed in the excluded medication. All concurrent therapies, including medications and supportive therapy (e.g., intubation, dialysis, and blood products), should be recorded from the date the patient is enrolled into the study through 3 months after completing TC-510 T cell therapy. After 3 months post TC-510 T cell infusion, only targeted concomitant medication will be collected, including immunosuppressive drugs, anti-infective drugs, vaccinations, and any therapy for the treatment of the patient’s malignancy for 1 year beyond disease progression. Specific concomitant medication collection requirements and instructions are included in the case report form (CRF) completion guidelines.

[00463] Prohibited Concomitant Medications

[00464] See the exclusion criteria for a detailed list of prohibited concomitant medications.

• In general, medications that might interfere with the evaluation of the investigational product should not be used unless absolutely necessary. Medications in this category include (but are not limited to):

• Immunosuppressive agents

• Corticosteroid anti-inflammatory agents including prednisone, dexamethasone, solumedrol, and cyclosporine. Immunosuppressive doses of systemic corticosteroids. Participants are permitted the use of topical, ocular, intra-articular, intranasal, and inhalational corticosteroids (with minimal systemic absorption). Adrenal replacement steroid doses > 10 mg daily prednisone are permitted. A brief (less than 3 weeks) course of corticosteroids for prophylaxis (e.g., contrast dye allergy) or for treatment of non-autoimmune conditions (e.g., delayed-type hypersensitivity reaction caused by a contact allergen) is permitted.

• Any concurrent systemic anti -neoplastic therapy (i.e., chemotherapy, hormonal therapy, immunotherapy, radiation, or standard or investigational agents for treatment of the disease under study), except as needed for treatment of disease progression.

• Any complementary medications (e.g., herbal supplements or traditional Chinese medicines) intended to treat the disease under study. Such medications are permitted if they are used as supportive care.

• Any live/attenuated vaccine (e.g., varicella, zoster, yellow fever, rotavirus, oral polio and MMR) during treatment and until 100 days post last dose.

[00465] Study Restrictions: Contraception

[00466] There are no data regarding the safety of TC-510 T cell during pregnancy or lactation in humans. Female patients who are pregnant, intending to become pregnant, or breast feeding are excluded from this study.

[00467] Female and male patients of reproductive potential must agree to avoid becoming pregnant or impregnating a partner, respectively. The required duration of contraception is described below:

• Female patients of childbearing potential must agree to use an effective method of contraception starting at the first dose of chemotherapy for at least 12 months thereafter and 4 months after the TC-510 T cell gene modified cells are no longer detected in the blood. • Male patients must agree to use an effective method of contraception starting at the first dose of chemotherapy and for 4 months thereafter or longer (if indicated in the country specific monograph/label for cyclophosphamide).

[00468] Female patients of childbearing potential are defined as premenopausal and not surgically sterilized.

[00469] Effective methods of contraception include intra-uterine device, injectable hormonal contraception, oral contraception, or 2 adequate barrier methods (e.g., diaphragm with spermicide, cervical cap with spermicide, or female condom with spermicide [spermicides alone is not an adequate method of contraception]).

[00470] Abstinence (relative to heterosexual activity) can be used as the sole method of contraception if it is consistently employed as the patient’s preferred and usual lifestyle and if considered acceptable by local regulatory agencies and IRBs. Periodic abstinence (e.g., calendar, ovulation, sympto-thermal, post-ovulation methods, etc.) and withdrawal are not acceptable methods of contraception.

[00471] Toxicity Management: Patients should be monitored and/or treated for toxicities, including the following: infection with Pneumocystis Jiroveci pneumonia, herpes virus, varicella zoster, and fungal infections; tumor lysis syndrome; cytokine release syndrome (CRS); fever and neutropenia; low hemoglobin or platelet count; any new onset neurotoxicity. Patients should receive prophylaxis for infection with Pneumocystis Jiroveci pneumonia, herpes virus, varicella zoster, and fungal infections according to National Comprehensive Cancer Network guidelines or standard institutional practice. All patients with significant tumor burden and without a contraindication should receive tumor lysis syndrome prophylaxis (e.g., allopurinol) as per institutional guidelines prior to TC-510 infusion. Prophylaxis should be discontinued when the risk of tumor lysis has passed.

Study Procedures and Schedule of Events

Clinical Assessments and Procedures

[00472] Demographics, medical history, and disease history will be collected and recorded. Physical examination, measurement of vital signs, performance status using ECOG performance scale, clinical safety assessments, laboratory assessments, and cardiac assessments will all be performed.

Tumor Response Assessments

[00473] Imaging scans of the chest, abdomen, and pelvis will be performed at leukapheresis eligibility, baseline, week 4, week 8, week 12, week 24, and every 12 weeks until confirmed disease progression, study completion, or withdrawal. Acceptable imaging modalities for this study include:

• Diagnostic-quality CT scan with oral and/or iv iodinated contrast of the chest and abdomen/pelvis (CT is the preferred modality for tumor assessments) • MRI of the abdomen/pelvis acquired before and after gadolinium contrast agent administration and a non-contrast enhanced CT of the chest, if a patient is contraindicated for contrast enhanced CT.

[00474] In addition to CT scans and/or MRIs, patients will undergo PET scans of the chest, abdomen, and pelvis at baseline, Week 4, Week 12, and as clinically indicated thereafter, as well as at time of disease progression, study completion, or withdrawal from the study. Tumor assessments will be evaluated according to the RECIST vl. l. (or mesothelioma specific RECIST criteria, if applicable). To allow time for an immune response to become apparent and to account for potential post-treatment transient inflammation of the tumor site (“pseudoprogression”), response assessments will not be carried out before 4 weeks post TC-510 infusion, unless there is unequivocal clinical evidence of deterioration. If disease progression is equivocal, confirmation of disease progression may be required by a follow-up scan performed at least 4 weeks apart, unless there is an immediate medical need to initiate anti-cancer therapy before the confirmatory scan can be performed. Disease progression will not be declared until results from the confirmatory scan are available. If confirmed, the date of progression will be that of the initial scan where progression was first suspected (i.e., not the confirmatory scan).

[00475] For clinical decision making, investigators will assess tumor response according to RECIST vl. l (or mesothelioma-specific RECIST criteria, if applicable). To the extent that it is feasible, local tumor assessments should be performed by the same radiologist. Additionally, scans will be submitted to a central imaging facility for independent read and to give a RESIST criteria response. Results from both reads will be summarized and reconciled at the end of the study phase.

[00476] For patients who have new lesions, response by RECIST (Nishino et al, 2013) will be assessed by the Investigator for exploratory purposes. For new lesions, information on whether the lesion is measurable or non-measurable will be recorded in the CRF.

[00477] Study images will be sent to a designated central vendor for assessment of tumor response. Review and interpretation of tumor response according to RECIST vl.l (or mesothelioma-specific RECIST criteria, if applicable) will be conducted by an appropriately qualified, trained, and experienced independent radiologist in accordance with an imaging review charter. The imaging review charter will also describe the procedures for CT/MRI and PET data handling after the images have been received by the central vendor from the sites.

[00478] TC-510 T Cell Retreatment (Applicable During Phase 2)

[00479] Patients treated in the phase 2 portion of the study who have a confirmed response (i.e., PR or CR) or at least SD for more than 4 months (16 weeks) post-TC-510 infusion and have relapsed will have an option to receive a second treatment regimen, known as retreatment, consisting of a course of lymphodepleting chemotherapy followed by a second dose of TC-510, should their disease progress after the first infusion. The second dose of TC-510 can be administered at or below the RP2D. The decision to undergo a second TC-510 regimen will be made by the treating Investigator and upon consultation and agreement of the TCR2 Therapeutics Medical Monitor. Patients will be required to meet the original treatment eligibility criteria again (including adequate MSLN expression) and should not have received any other therapy for their underlying malignancy. The second dose will be administered following the same procedural requirements as the first dose, including the post-treatment study requirements. Patients meeting treatment eligibility criteria for a second dose may proceed to TC-510 infusion no sooner than 4 months and no later than 1 year (52 weeks) following completion of the first TC-510 dose.

[00480] Patients eligible for retreatment should be reconsented following discussion regarding benefits and risks of TC-510 therapy and if applicable, a careful explanation about the need to undergo leukapheresis a second time for the manufacturing of TC-510 prior to performing any study related procedures or treatment. This conversation should be recorded in the patient’s source document. Correlative Studies and Research Assessments

[00481] Correlative studies and research assays will be performed during the trial to monitor the biological parameters that may impact TC-510 T cell treatment outcome. Such studies include assessing phenotype, function, and persistence of the infused TC-510, cytokine measurements, and immunogenicity studies, among others. Studies will be conducted on tumor biopsies, PBMCs, serum or plasma.

[00482] Research studies conducted on blood samples, manufactured TC-510 product, ascites and serosal effusions from patient samples may include the following exploratory endpoints, among others:

• Analysis of PBMC’s and TC-510 phenotype and persistence by flow cytometry

• Determination of TC-510 persistence using PCR-based assays

• Characterization of PBMCs or TC-510 (pre- and post-infusion) using molecular or functional assays to assess cytotoxicity, cytokine release, proliferative capacity, or gene expression profiles

• Assessment of cytokine release by TC-510 using multiplexed assays to measure proinflammatory cytokines

• Detection of anti-TC-510 antibody levels pre- and post-infusion using enzyme-linked immunosorbent assay (ELISA)

• Quantitation of circulating tumor cells and circulating tumor DNA pre- and post TC-510 infusion

[00483] Biopsy research studies may include, among others: • Tissue expression of the MSLN antigen and PD-L1 by IHC

• Detection of tumor-infiltrating TC-510 post-infusion using RNAscope technology and/or PCR

• Imaging analysis to quantify and phenotype immune cell infiltrates by multiplexed IHC and immunofluorescence

• Gene expression profiling

[00484] If a patient has an adverse event, an additional biopsy (e.g., skin, ascites, serosal fluid, gastrointestinal tract, bone marrow, tumor) or blood (serum and PBMC) samples may be requested, and studies may be performed on those samples aimed at understanding the underlying pathophysiology of the ongoing adverse event.

[00485] Plasma is collected at baseline and at select visits post-infusion, to allow for measurement of cytokines in the blood. Plasma is also collected from patients with suspected CRS with samples being taken approximately every 24-48 hours until symptoms improve or an alternative diagnosis is confirmed. Cytokines, growth factors and soluble receptors including but not limited to IL-ip, IL-6, IFN-y TNF-a, IL-2, IL-8, IL-10, IL-12, IL-13, IL-15, and GM-CSF will be measured utilizing a multiplexed assay and will be performed following Good Laboratory Practice procedures. All other measurements may be performed as exploratory.

[00486] Plasma samples will also be used to detect the presence of anti-MHl antibodies prior to infusion and at week 8 post-infusion. For plasma samples that demonstrate positivity of anti-MHl human antibodies at the week 8 visit, an attempt should be made to obtain and test additional plasma samples at week 12, week 24, and at 12-week intervals thereafter until the antibody levels return to baseline (or become negative) or up to 1 year from date of TC-510 infusion, whichever occurs first.

[00487] Tumor Biopsies'. The activity of TC-510 will be impacted by other cellular elements within the tumor microenvironment (e.g., regulatory T cells). Evaluating the “immune landscape” within the tumor pre- and post-infusion of TC-510 may provide valuable insight into characteristics of the tumor microenvironment that influence clinical outcomes. For this reason, core needle biopsies will be requested at 4 specific time points:

• Pre-screening: archival tissue biopsy may be used for pathological confirmation of diagnosis and MSLN expression determination if obtained within the 12 months preceding the prescreening ICF date. If adequate archival tissue is unavailable, a fresh tissue biopsy should be obtained, in which case, such fresh biopsy can also be used as the baseline biopsy prior to initiation of study treatment (i.e., lymphodepleting chemotherapy).

• Baseline: a fresh tissue biopsy will be obtained (if not obtained at pre-screening) for the conduct of translational studies to evaluate the immune status of the tumor prior to TC-510 infusion, as described herein, including, among others: T cell infiltration, presence of immune activation/exhaustion markers, and tissue expression of immune checkpoints.

• MSLN and PD-L1 expression analysis will also be assessed on the fresh tumor biopsy prior to study treatment. If a fresh tissue biopsy is obtained for screening purposes and the remaining tissue is sufficient for research studies, the baseline biopsy can be omitted. Otherwise, the baseline biopsy material may be collected anytime between 2 months and 1 week prior to the start of lymphodepleting chemotherapy, favoring a time point closer to the time of TC-510 infusion. In the event a fresh biopsy is performed at baseline and the patient experiences unforeseen delays in dosing, the window for the fresh biopsy may be extended with approval from the Sponsor.

• Week 8 (± 7 days): a fresh tissue biopsy will be obtained at this time point, which is when an active anti -tumor response by the infused TC-510 is expected. Translational studies will be conducted on this biopsy to evaluate TC-510 infiltration and the immune status of the tumor upon TC-510 infusion as described in section 9.6, MSLN and PD-L1 expression will also be assessed on this tumor biopsy for research purposes.

• After confirmation of disease progression (± 2 weeks): a fresh tissue biopsy will be obtained at this time point (unless tumor is not safely accessible) to evaluate the immune status of the tumor upon TC-510 infusion, as described herein, and mechanisms of immune escape (e.g., target antigen downregulation, T cell immune exclusion). Ideally, biopsy material obtained after confirmation of disease progression should be collected from lesions that have progressed and from new lesions to best address mechanisms of resistance and to determine eligibility for retreatment.

• Autopsy Tissue Collection: In the event a patient dies, and an autopsy is performed upon request of the participant’s family or next of kin, study investigators may ask permission from the participant’s family to collect tissue during the autopsy for research purposes. This may help investigators learn about the effects of treatment with genetically modified cells. Subject to the consent of the participant’s family, the Sponsor may use tissue collected during the autopsy for future testing. The Sponsor will only request tissue if an autopsy is requested by the participant’s family, the Sponsor will not require an autopsy to be performed solely for the purpose of the study.

• Retreatment Baseline: the same translational studies as for the initial Baseline biopsy will be conducted to evaluate the immune status of the tumor prior to TC-510 retreatment. MSLN and PD-L1 expression analysis will also be assessed on the fresh tumor biopsy prior to retreatment (i.e., lymphodepleting).

[00488] When possible, biopsies should consist of multiple cores taken from more than one lesion. In cases where tumor lesions may not be amenable to core needle biopsy, fine needle aspirates may be obtained based on interventional radiology recommendations. Tumor tissue should either be taken from non-target lesions or from target lesions > 2 cm. Every attempt should be made to obtain biopsies at all time points from the same lesion(s). The radiological (or clinical) status of the lesion(s) biopsied should be noted at the time (e.g., decreased, stable, increased size or activity). In patients who have ascites and/or serosal effusion, should there be a clinical requirement for removal of the effusion fluids at any time during study, samples are requested to be collected for the conduct of research studies. When available, ascites, serosal effusion fluids, and lumbar fluid, should be collected in addition to, and not instead of the requested tumor biopsies, with the exception of MPM cases where tumor biopsy is not accessible and/or available. Ascites, serosal effusion, and lumbar fluid specimens will be used to interrogate the tumor microenvironment prior to and after TC-510 infusion to address mechanisms of sensitivity or resistance to therapy as well as kinetics of tumor clearance.

[00489] TC-510 persistence'. The presence, expansion, and persistence of TC-510 will be monitored in peripheral blood by both quantitative PCR and flow cytometry.

[00490] TC-510 T cells phenotype and activity. A range of assays will be performed to elucidate the phenotype and activity of the infused TC-510 including (but not limited to): immunophenotyping of TC-510 in manufactured cell product prior to infusion and in the blood (and tumor, when available) post-infusion by flow cytometry to assess differentiation and activation status; detection of antibody responses against the anti-MSLN MHl binder of TC-510; and ex -vivo activity of PBMCs and/or TC- 510 pre- and post-infusion to assess T cell functionality by cytotoxicity, cytokine release, proliferative capacity, or molecular characteristics.

[00491] Liquid Biopsies: The presence of circulating tumor cells (CTCs) will be assessed as an early response biomarker in whole blood at baseline and up to 4 time points post TC-510 infusion. Tumor cells in circulation will be measured by immunofluorescence-based assays. Additionally, expression of MSLN in CTCs and TC-510 in the white blood cell compartment will be assessed in the same assay. In addition to CTCs, presence of circulating tumor DNA (ctDNA) will be assessed in plasma samples as an early response biomarker at baseline and up to 4 time points post TC-510 infusion.

Dose Escalation

[00492] The objective of the dose-escalation phase of the study (phase 1) will be the evaluation of DLTs and the determination of the RP2D. If the MTD (defined as the dose administered at 1 dose level below the dose in which DLTs were observed in > 33% of patients) is determined during the dose escalation phase, then the MTD will be the recommended RP2D. See Table 2 above for the TC-510 dose levels.

[00493] Each patient will receive a single dose of TC-510. At each dose, TC-510 will be given to at least 3 patients following lymphodepleting chemotherapy (fludarabine 30 mg/m ² /d on days -7 through -4 and cyclophosphamide 600 mg/m ² /d on days -6 through -4). Patients will be enrolled at the dose levels (DL) shown in Table 2 to determine the RP2D.

[00494] A variation on the target dose of 15% (i.e., ± 15%) will be allowed at each dose level. A planned stagger of 28 days will be instituted between the first patient infused at each dose level and the 2nd patient.

[00495] Patients 2 and 3 in any given cohort may be infused simultaneously.

[00496] In the event of excessive toxicity at the initial dose level (DL0), the study will resume at DL- 1 (i.e., 25 x io ⁶ transduced T cells on day 0). Dose escalation will be directed by the SRT upon review of the safety data.

[00497] Once identified, up to 10 patients may be subsequently treated at the RP2D to further delineate the toxicity profile of TC-510 prior to advancing to the phase 2 portion of the study.

[00498] For the purpose of safety onboarding, a 14-day stagger will be implemented for the first 3 patients treated at each new site (i.e., with no prior experience administering TC-510).

[00499] In the event the RP2D is determined for one or more specific indications, dose escalation may continue further to determine the indication-specific RP2D for other indications where TC-510 may be tolerated at higher doses. Once the RP2D for a specific indication is determined, enrollment of the Phase 2 specific arm for such indication may commence.

[00500] Protocol stagger, safety observation and dose escalation rules will continue to apply to patients within the indication-specific dose escalation cohort(s) until the indication-specific RP2D is determined.

[00501] The sample size of the RP2D cohort may be further expanded to include 6 additional patients (at the discretion of the SRT) in order to better characterize safety prior to launching the phase 2 of the study. There will be no requirement to stagger patients during this portion of the study. The SRT may declare the RP2D at any time based on available safety data independent of whether the MTD has been reached or not.

Example 3: TFP Constructs and PD-1 Fusion Proteins

[00502] Examples of the anti-MSLN TFP constructs generated include anti-MSLN-linker-human CD3s chain (including extracellular, transmembrane, and intracellular domains), with the anti-MSLN antigen binding domain being the scFv or sdAb with sequences disclosed in Table 6. [00503] Primary T cells were activated and transduced with a lentiviral construct containing (i) the anti-MSLN TRuC sequence (MSLN VHHDNA fragment linked to a CD3 epsilon DNA by a linker sequence), and (ii) a sequence encoding the PD-lxCD28 switch receptor (extracellular PD-1 domain, transmembrane PD-1 domain, and intracellular signaling domain of CD28); (SEQ ID NO: 58). The anti-MSLN TRuC sequence is linked to the PD-lxCD28 switch receptor sequence by a linker comprising a protease cleavage site (T2A). pRLPC was used as the lentiviral vector; various other vectors may be used to generate fusion protein constructs (e.g., pLRPO, pLCUS, or pLKaUS). Source of TCR Subunits

[00504] Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain. The CD3 epsilon, CD3 delta, and CD3 gamma subunits have an intracellular domain. A human TCR complex contains the CD3 -epsilon polypeptide, the CD3- gamma polypeptide, the CD3-delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide. TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide. The human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766. The human CD3- gamma polypeptide canonical sequence is Uniprot Accession No. P09693. The human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234. The human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963. The human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1. The human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.

[00505] The human CD3-epsilon polypeptide canonical sequence with signal peptide is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQ H NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCEN C MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPP VPNPDYEPIRKGQRDLYSGLNQRRI.

[00506] The signal peptide of human CD3s is:

[00507] MQSGTHWRVLGLCLLSVGVWGQ.

[00508] The human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGK M IGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFA E IVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN . [00509] The signal peptide of human CD3y is:

[00510] MEQGKGLAVLILAIILLQGTLA. [00511] The extracellular domain of human CD3y is:

[00512] QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSN

AKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS.

[00513] The transmembrane domain of human CD3 y is:

[00514] GFLFAEIVSIFVLAVGVYFIA.

[00515] The intracellular domain of human CD3y is:

[00516] GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN.

[00517] The human CD3-delta polypeptide canonical sequence is:

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITR LDLGKRI

LDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFA GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS.

[00518] The signal peptide of human CD36 is:

[00519] MEHSTFLSGLVLATLLSQVSP.

[00520] The extracellular domain of human CD36 is:

[00521] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYK

DKEST VQVHYRMCQ SCVELDP AT VA.

[00522] The transmembrane domain of human CD36 is:

[00523] GIIVTDVIATLLLALGVFCFA.

[00524] The intracellular domain of human CD36 is:

[00525] GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK.

[00526] The human CD3-zeta polypeptide canonical sequence is:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSR SADAPA

YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA E AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[00527] The human TCR alpha chain canonical sequence is:

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGL DSPI

WFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSR STQP MHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSP AT TTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYP TC PAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA.

[00528] The human TCR alpha chain C region canonical sequence is:

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMD FKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFR ILLL K V AGFNLLMTLRLW S S . [00529] The human TCR alpha chain human IgC sequence is:

[00530] PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL S.

[00531] The transmembrane domain of the human TCR alpha chain is:

[00532] VIGFRILLLKVAGFNLLMTLRLW.

[00533] The intracellular domain of the human TCR alpha chain is:

[00534] SS

[00535] The human TCR alpha chain V region CTL-L17 canonical sequence is:

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFD YFL

WYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCA AKGAGT ASKLTFGTGTRLQVTL.

[00536] The human TCR beta chain C region canonical sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS TDPQ

PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF.

[00537] The human TCR beta chain human IgC sequence is:

[00538] EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGV

STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDR AKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE.

[00539] The transmembrane domain of the human TCR beta chain is:

[00540] ILLGKATLYAVLVSALVLMAM.

[00541] The human TCR beta chain V region CTL-L17 canonical sequence is:

MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQT LGQ

GPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAG LNQPQ HFGDGTRLSIL.

[00542] The intracellular domain of the human TCR beta chain is:

[00543] VKRKDF.

[00544] The human TCR beta chain V region YT35 canonical sequence is:

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQT MMR GLELLIYFNNNVPIDD SGMPEDRF S AKMPNASF STLKIQPSEPRDS AVYFC AS SF STC S ANYG YTFGSGTRLTVV.

[00545] The human TCR gamma chain C region canonical sequence is: [00546] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQ EGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPK D NCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS.

[00547] The human TCR beta gamma human IgC sequence is:

[00548] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQ EGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPK D NCSKDANDTLLLQLTNTSA.

[00549] The transmembrane domain of the human TCR gamma chain is:

[00550] YYMYLLLLLKSVVYFAIITCCLL.

[00551] The intracellular domain of the human TCR gamma chain is:

[00552] RRTAFCCNGEKS.

[00553] The human TCR delta chain C region canonical sequence is:

[00554] SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAV KLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH T EKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL.

[00555] The human TCR delta human IgC sequence is:

[00556] SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAV KLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVH T EKVNMMSLTV.

[00557] The transmembrane domain of the human TCR delta chain is:

[00558] LGLRMLFAKTVAVNFLLTAKLFF.

[00559] The intracellular domain of the human TCR delta chain is: L.

Expression Vectors

[00560] In some embodiments, TFP constructs are in a vector that further contains a sequence encoding a PD-lxCD28 switch. The PD-lxCD28 switch may be encoded in the same open reading frame as the TFP, and separated by a self-cleaving peptide (e.g., a P2AW or a T2A self-cleaving peptide).

[00561] Expression vectors are provided that include: a promoter (eukaryotic elongation factor 1 alpha (EFla promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).

[00562] The TFP- and PD-1 fusion protein-encoding nucleic acid constructs were cloned into a lentiviral expression vector as is described above. The lentiviral transfer vectors were used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles. HEK293 cells (e.g., suspension adapted HEK293 cells, e.g., Expi293F-cells) were incubated with the transfer DNA plasmid, Gag/Pol plasmid, Rev plasmid, and VSV-G plasmid. After incubation, lentivirus containing supernatant was collected, centrifuged and filtered, and used for infection or aliquoted and stored at -80°C for future use.

Example 4: TRuC T Cell Enhancement with PD-lxCD28 Switch Receptor

[00563] Methods section

[00564] The following are examples of methods used in this study examining TRuC T cell enhancement with a PD-lxCD28 switch receptor mechanism.

[00565] MSLN-targeting e-TRuC was generated as described in (Ding et al.). A PD1TM switch receptor was generated by isothermal assembly of the ecto- and transmembrane domains of PD-1 (QI 5116 amino acids 1-191) to the intracellular domain of CD28 (Pl 0747 amino acids 180-220). Similarly, a CD28TM switch receptor was generated by isothermal assembly of the ectodomain of PD-1 (Q15116 amino acids 1-170) to the transmembrane and intracellular domains of CD28 (P10747 amino acid 153-220). MSLN-targeting e-TRuC and the switch receptor were cloned on the same lentivirus expression vector upstream and downstream of a T2A signal, respectively. For the generation of target cell lines, full length firefly luciferase (FLuc) or the PD-L1 ecto- and transmembrane domains was cloned into pCDH-CMV-MCS-EFla-Neo. Full length human MSLN (MSLNFL) was cloned into pCDH- pCDH-EFla-MCS-T2A-Puro (SBI, Palo Alto, CA), using Xbal and EcoRI restriction sites.

[00566] Lentivirus was prepared by transient transfection of HEK293 suspension cells maintained in FreeStyle™ 293 Expression Medium (ThermoFisher Scientific, Waltham, MA). The lentiviral vector and packaging plasmids (pCMV-co. Gag/Pol, pCMV-co. VSV-G, pRSV-co.REV; were mixed with PEIpro (Polyplus Transfection, New York, NY) and added to suspension cultures of HEK293 cells. After medium change at 24 hours post transfection, culture supernatant was collected at 48 hours post transfection. Supernatant was clarified by centrifugation at 3000 x g for 30 minutes followed by filtration with a 0.45 pm PES filter (VWR, Franklin, MA) and precipitation with Lenti-X virus concentrator (TakaraBio, Japan). Clarified supernatant was resuspended in TexMACS medium (Miltenyi Biotech, Germany) supplemented with 3% human AB serum (Gemini Bio-Products, West Sacramento, CA), aliquoted, and stored at -80°C until use. The infectious titer of the lentivirus (JU/mL) was estimated by quantitating the number of integrated lentiviral vector copies (VCN) per genome of infected HEK293T cells (CRL-1573™, ATCC, Manassas, VA) with real-time PCR using primer/probe set targeting the OPRE region of the lentiviral vector. [00567] Tumor cell lines were purchased from ATCC (MSTO-211H [CRL-2081™]; Mannasas, VA) or Millipore Sigma (A2780 [C30]; St Louis, MO). Cells overexpressing Luc, PD-L1 ectodomain, and/or FL MSLN were generated from the parental cell lines with lentivirus and stably transduced cells were selected with neomycin (Millipore Sigma) and/or puromycin (Corning, Bedford MA). MSTO-FL MSLN-Luc and MSTO-FL MSLN-PD-Ll-luc co-expressing cells were generated from the corresponding parental cell line with lentivirus (as described above). Stably transduced cells were selected with neomycin in combination with puromycin (FL MSLN, Luc, and PD-L1 co-expressing cells).

[00568] Primary human T cells were sorted from a leukapheresis product (Hemacare, Van Nuys, CA) by magnetic bead separation using anti-CD4 and anti-CD8 microbeads according to the manufacturer’s protocol (Miltenyi Biotech). T cells were activated using T Cell TransAct, Human (Miltenyi Biotech) at 1 : 1 ratio and cultured in TexMACS medium (Miltenyi Biotech) with 3% human AB serum (Gemini Bio-Products) and 12.5 ng/mL of human IL-7 and 12.5 ng/mL of human IL-15 (Miltenyi Biotech). T cell transduction was carried out 24 hours after activation at an MOI of 1-5 in the presence Lentiboost (Sirion Bitotech GmbH, Germany). Transduced T cells were expanded by passaging in TexMacs for 10 days and frozen in Bambanker media (Wako Chemicals, USA) prior to use in functional assays.

[00569] The transduction efficiency of engineered T cells as well as the in vitro expansion, activation/exhaustion, and proliferation were analyzed by flow cytometric analysis. Cells were stained using fluorescently labeled antibody cocktails and data were acquired on the BD LSR Fortessa™ X-20 cell analyzer. Data analysis was performed with the FlowJo software (TreeStar Inc, Ashland OR). Detailed methods are provided in the supplemental material.

[00570] For the luciferase activity based tumor cell cytotoxicity assay, luciferase-expressing tumor cells were plated in triplicates in a 96-well plate at 10,000 cells per well and T cells were added at the desired effector-to-target (E-to-T) ratios. After 24-hour co-culture, 50% of the culture supernatant was removed for cytokine analysis. Cell viability was determined using the Bright-Glo™ Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s protocol. Relative luminescence unit (RLU) was measured using the SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). The percentage of tumor cell killing was calculated by the following formula: % tumor cell lysis = 100% x (1 - RLU (tumor cells + T cells)/RLU (tumor cells)).

[00571] For TRuC-T cell coculture assays with target cell lines, TRuC-T cells were first thawed and rested in IL-2 (300 U/mL) for 72 hours. At the end of the rest period, TRuC-T cells were then normalized for transduction efficiency, and then plated in a 96-well U-bottom plate at a 1 : 1 ratio with 1.0e5 Streck-treated tumor cells (Streck, USA) for up to 96 hours. Culture supernatants were harvested from replicate plates at 24 or 72 hours and stored at -80°C until sample analysis. Detailed methods, including rechallenge assays conditions, provided in the supplemental material.

[00572] For stimulation and intracellular detection of ERK phosphorylation, cryopreserved and freshly thawed effector non-transduced T-cells, TC-210 TRuC T-cells, and TC-210 TRuC T-cells transduced with PD1 *CD28 switch receptor were co-cultured with target cell lines MSTO, MSTO- MSLN, or MSTO-MSLN-PDL1 at a 1 : 1 ratio (3 x 10 ⁵ each) in 96 well round bottom plates in RPMI 1640 at 37°C. Effector cells treated with PMA (60 pg/mL) and ionomycin (1 pg/mL) served as a positive control for stimulation of ERK. Stimulation was achieved by mixing target and effector cells, followed by centrifugation at 300 x g for 5 minutes at 37°C. Reactions were quenched by 1 : 1 dilution into 4 °C PBS containing 8% formaldehyde and incubation at 4 °C for 30 minutes. Following centrifugation, cells were washed then suspended in methanol prior to cell staining and flow cytometric analysis. Cells were stained with anti-camelid VHH-Af488 fusion (Genscript cat. A01862), anti-CD3-BV605 fusion (Biolegend cat. 300459), anti-PD-l-PE fusion (Biolegend cat. 329905), anti-phospho-p44/42 MAPK (Erkl/2) (Thr202/Tyr202)-PE-Cy7 fusion (Cell Signaling Technology cat. 98168s). Staining was visualized by flow cytometry and population gating was determined from FMO controls.

[00573] For the s.c. xenograft model, 1 x 10 ⁶ MSTO-FL MSLN-PD-Ll-Luc cells were resuspended in sterile PBS, mixed 1-to-l with ice cold Matrigel® (Coming, Tewksbury, MA) and then injected subcutaneously in the dorsal hind flank of 7-8 week old female class Eclass II negative NSG mice (NOD.Cg-Prkdc ^scid H-2Kl ^tmlBpe H2-Abl ^emlMvw H2Dl ^tmlBpe I12rg ^tmlwj l/SzJ) from the Jackson Laboratory (Bar Harbor, ME). Engineered human T cells were administered at a dose of 2.0 x 10 ⁶ TRuC+ T cells per mouse, via tail vein injection when the tumor size was between 150-200 mm ³ . Mice were randomized into treatment groups by tumor burden prior to injection of human T cells. Tumor growth was monitored as tumor volume by caliper measurement and IVIS imaging twice weekly. The volume of tumor was calculated as: tumor volume = (length x width ² )/2. For tumor rechallenge, MSTO-MSLN-PD-Ll-Luc cells were prepared as described above and injected subcutaneously in the opposing flank.

[00574] Results section

[00575] The objective of this study was to functionally compare two versions of a PD-lxCD28 switch receptor (PD1TM and CD28TM) that differed in their transmembrane (TM) domain and evaluate potential improvements in T cell receptor fusion construct (TRuC) T cell function and persistence. [00576] Purified T cells were activated and transduced with a lentiviral construct expressing only an anti-MSLN TRuC (TC-210), or the anti-MSLN TRuC in tandem with a PD-lxCD28 switch receptor with PD1 or CD28 transmembrane domain (PD1TM or CD28TM respectively; FIG. 5A). After a 9- day expansion following transduction, a similar transduction efficiency, assessed as total percent TRuC ⁺ T cells, was observed between the TC-210 alone and TC-210 + PD-lxCD28 switch receptor groups, while the CD28TM group showed significantly lower transduction efficiency compared to PD1TM group (FIG. 5C). Both TC-210 + PD-lxCD28 switch receptor groups showed a statistically minor reduction in median fluorescence intensity (MFI) of TRuC ⁺ expression compared to TC-210 alone (FIG. 5D). Both PD1TM and CD28TM groups displayed similar levels of PD-1 expression, indicating comparable levels of receptor expression, which were ~18-fold higher than endogenous PD-1 levels in the TC-210 alone (FIG. 5E). The ratio of CD4 ⁺ to CD8 ⁺ T cells was significantly increased in all transduced groups in comparison to non-transduced (NT) controls, and a further increase was observed in the CD28TM group compared to TC-210 alone, and to the PD1TM group (FIG. 5F). All the T cell products showed comparable profiles with respect to memory phenotype (FIG. 11 A), activation marker expression, and inhibitory marker expression (FIGs. 11B-C).

[00577] The anti-tumor response of TC-210 and TC-210 + PD I CD28 switch receptor T cells was assessed using a mesothelioma cell line, MSTO-211H, engineered to express human MSLN (MSTO- MSLN) or MSLN and PD-L1 (MSTO-MSLN-PDL1). Resting TRuC T cells were co-cultured with the MSLN-negative cell line A2780 (C30), MSTO-MSLN, or MSTO-MSLN-PDL1 and cytotoxicity was assessed after 24-hours. Potent, antigen-specific cytotoxicity against both MSTO-MSLN and MSTO-MSLN-PDL1 was observed for TC-210, PD1TM, and CD28TM groups, with no observable differences in tumor lysis, irrespective of PD-L1 expression by the target cells (FIG. 6A). Both PD- lxCD28 switch receptor groups displayed higher levels of Thl cytokine production (IL-2, TNF-a, and GM-CSF) than TC-210 alone against both MSTO-MSLN and MSTO-MSLN-PDL1 cells (FIG. 6B). There was a moderate level of endogenous PD-L1 expression by the MSTO-MSLN cell line (FIG. 12). To confirm that increased cytokine production was being mediated by PD-lxCD28 switch receptor engagement by PD-L1, a PD-1 blocking antibody was added to the co-culture conditions. Addition of the PD-1 blocking antibody reduced Thl cytokine production in the PD1TM and CD28TM groups to the levels observed for TC-210 alone for IL-2, GM-CSF, IFN-y, and TNF-a (FIG. 6C)

[00578] Phosphorylation of ERK (pERK) was measured in T cells transduced with TC-210 and the TC-210 + PD-lxCD28 switch receptor, following co-culture with MSTO, MSTO-MSLN, and MSTO-MSLN-PDL1 cells to evaluate TCR-associated signaling across treatment groups (FIGs. 6D- H). TRuC T cells cultured with MSTO cells produced little pERK signaling by 10 minutes (FIGs. 6E-F). The frequency of MSLN TRuC T cells expressing pERK increased by 4-6 fold upon culture with MSTO-MSLN or MSTO-MSLN-PDL1 (FIG. 6E), with PD1TM group modestly expressing the highest level of pERK at this early timepoint (FIG. 6F). At 60 minutes of co-culture, pERK expression was significantly greater for PD1TM T cells stimulated with MSTO-MSLN or MSTO- MSLN-PDL1 target cells than for CD28TM or TC-210 TRuC T cells (FIGs. 6G-H).

[00579] To assess sensitivity of the PD-lxCD28 switch receptor to PD-L1, increasing concentrations of plate-bound Fc-conjugated PD-L1 in the presence of a fixed concentration (1.0 pg/mL) of platebound MSLN was applied to determine regulation of the TRuC T cells by PD-L1 (FIG. 13A). PD- L1 alone did not induce cytokine production by any TRuC T cell group and in the absence of PD-L1, MSLN antigen alone induced comparable levels of cytokine production by all treatment groups (FIG. 7A). Dual stimulation with both MSLN and PD-L1 elicited increased levels of cytokine production relative to TC-210, with the CD28TM groups producing significantly higher levels of IFN-y, even at the lowest concentration of PD-L1 (FIG. 7A; TC-210: 1433.5 pg/mL, PD1TM: 3247.6 pg/mL, and CD28TM: 23,000 pg/mL). IL-2 production did not differ statistically between the PD1TM and the CD28TM groups, although the CD28TM group produced significantly more IL-2 than TC-210 at the highest concentration of PD-L1 tested. The PD1TM group showed a clear dosedependent response to plate-bound PD-L1, while the CD28TM group was strongly activated even at low PD-L1 levels. After 96 hours of culture, the fold-expansion of the CD28TM group was observed to peak at 2.0 pg/mL of PD-L1 and then decreased at higher concentrations, whereas the PD1TM group continued to expand at the higher concentrations of PD-L1 (FIG. 13B). The CD28TM group also showed a decrease in viability, measured as percentage of viable CD3 ⁺ cells, with increasing PD-L1 concentrations (FIG. 13C). TRuC T cell groups were cultured with the MSLN negative cell line C30 (C30-PDL1), and parental MSTO-211H cells (MSTO-PDL1) to examine activation thresholds of the PD-lxCD28 switch receptors. When cultured with C30 or C30-PDL1 cell lines, only a baseline response was observed in each of the TRuC T cell cultures groups (FIG. 7B). When cultured with the parental MSTO cell line, only the CD28TM group showed a significantly heightened cytokine response, which was reduced to baseline when PD-L1 was over-expressed (MSTO-PDL1).

[00580] To determine the relative contributions of PD1 competition and CD28 co-stimulation to the enhanced effector function of PD1TM TRuC T cells, antigen-induced pERK levels were measured in PD1TM T cells made non-functional through mutation or deletion of the CD28 signaling domain (FIG. 8A). One variant, the PDlTM ^Mutant , harbors mutations to the SH3 domains of the intracellular CD28 domain; these included binding sites for Lek, Grb2, and ITK (PYAPAAYAA; PRRPAARRA), the Tyr phosphorylation site required for PI3K-Akt signaling (Y191AA191), and a putatively phosphorylated serine (S189AA189) of unknown function in the signaling network (Tian et al. 2015). The other variant, the PDl ^Trunc , has only the membrane bound PD1 ectodomain and lacks a CD28 signaling domain. Both variants were expressed and showed positive expression for TRuC and staining for exogenous PD-1 receptor (FIG. 8B). No significant differences in ERK phosphorylation were observed between the treatment groups (FIG. 8C). Supernatants collected from a 72-hour coculture with tumor cells yielded limited cytokine secretion by both PDlTM ^Mutant and PD-l ^Trunc groups (FIG. 8D)

[00581] TRuC T cell groups were subjected to an in vitro tumor rechallenge assay with MSTO- MSLN-PDL1 tumor cells at a low effector-to-tumor ratio, followed by a rechallenge every 96 hours to assess the fitness of TRuC T cells. TRuC T cells normalized for transduction showed comparable expansion in response to the initial antigen exposure, with no discernible differences in TRuC fold expansion between TC-210, PDlTM ^Mutant and PD-l ^Trunc cultures (FIG. 9A). Following the second rechallenge on day 8, these cultures displayed a contraction relative to the day 4 peak. The PD1TM group (now referred to as TC-510 group) demonstrated an increase in the number of TRuC T cells and following a second round of stimulation, the TC-510 group showed a continued accumulation of TRuC T cells, expanding by 23-fold in comparison to TC-210 (FIG. 9A). Across the rechallenge assay, there were no significant differences in cytokine production between TC-210, PDlTM ^Mutant and PD-l ^Trunc groups (FIG. 9B). The TC-510 group produced a significantly higher level of IFN-y (4.6-fold) and IL-2 (78-fold) relative to TC-210 after the first round of stimulation. Culture morphological analysis performed prior to the third round of stimulation revealed that cells in the TC-210, PDlTM ^Mutant and PD-l ^Trunc groups displayed a more diffuse morphological pattern in comparison to the defined cell clusters in the PD1TM (TC-510) culture (FIG. 9C). Flow cytometric analysis showed the TC-210, PDlTM ^Mutant and PDl ^Trunc cultures displayed co-expression of the exhaustion markers TIGIT and LAG-3 by TRuC T cells, with no exhaustive phenotype acquired by the TC-510 group (FIG. 9D).

[00582] MHC Class I/II null NSG mice were subcutaneously (SC) implanted with MSTO-MSLN- PDLl-Luc cells on. Once the tumors were established (150-200 mm ³ ), tumor-bearing mice were sorted into treatment cohorts of n=10 mice and treated with 2.0 x 10 ⁶ TRuC T cells (TC-210 or TC- 510), non-transduced (NT) control T cells, Vehicle, or received no injection (Naive). NT and Vehicle groups displayed progressive tumor growth. Mice receiving TC-210 or TC-510 cells showed comparable anti -tumor activity with tumor shrinkage first evident on day 10 post-infusion and complete tumor clearance by day 17 (FIGs. 10A-B). A tumor rechallenge was performed on day 44 to assess long-term functional persistence, without retreatment of TRuC T cells. After initial tumor regrowth, mice previously treated with TC-210 or TC-510 were able to clear the tumors, however all TC-210 treated mice eventually experience tumor recurrence (FIGs. 10B). Recurrence was limited to 1/8 mice in the TC-510 group (FIG. 10B) and TC-510 mice that rejected the rechallenge tumors showed durable protection for the remainder of the observation period (244-days post TRuC T cell administration).

[00583] Discussion section

[00584] T cell receptor fusion construct (TRuC®) T cells can differentiate themselves from CAR-T cells by demonstrating faster tumor regression, lower cytokine production, increased tumor infiltration, increased oxidative metabolism and enhanced persistence. However, like native T cells, TRuC-T cells may remain susceptible to inhibition in the TME by the PD-1/PD-L1 axis. Furthermore, while engineered costimulatory signals may not be required for the in vivo efficacy of TRuC-T cells, in contrast to CAR-T cells, the delivery of costimulation in conjunction with TRuC activation may enhance T cell function and persistence. This example provides the engineering of a PDlxCD28 switch receptor that co-opts tumor PD-L1 expression to drive CD28 co- stimulation. These results translated to in vivo assessments, where the PDlxCD28 switch receptor in TRuC T cells supported antitumor results in mouse xenograft models.

[00585] Both blocking PD-1 and enhancing CD28 signaling can be attractive strategies for driving tumor-targeted T-cell activity and persistence in the TME. Indeed, the approved anti-PD-1 drugs may have significantly changed the standard of care treatment in multiple cancers, improving overall survival, progression free survival and durability of response (He and Xu 2020). PD-1 inhibition of T-cell signaling is mediated by recruitment of SHP phosphatases that deactivate TCR signaling by targeting key kinases of the TCR and CD28 signaling pathways (Arasanz et al. 2017; Gaud et al. 2018). Recent studies demonstrated that CD28 itself is a target of PD-1 -activated SHP phosphatases (Hui et al. 2017). Regardless of PD-1 -mediated inhibition, activation of CD28 by ligation to B7.1 and B7.2 on antigen-presenting cells can provide a costimulatory signal that gates and augments endogenous TCR signaling (Azuma et al. 1993; Freeman et al. 1989). CD28 phosphorylation can potentiate T-cell activation, leading to enhanced proliferation and effector function, notably induction of the TH1 cytokines IL-2, TNF-a, and IFN-y (Esensten et al. 2016; Fife and Bluestone 2008; Okkenhaug and Rottapel 1998; Rohrs et al. 2020).

[00586] The selection of the TM domain utilized in a switch receptor can have a profound effect on its function. The example provided herein functionally compares PDlxCD28 switch receptors utilizing either a PD1TM or CD28TM. While both versions of the switch receptor provided a CD28 signaling enhancement to TRuC-T-cells as evidenced by heightened cytokine secretion, Thl polarization, and increased TCR signaling versus TC-210, there were important functional differences between these two versions of the switch receptor. The data show that the PD1TM switch receptor provided a more favorable profile for efficacy and safety as it demonstrated a greater dynamic response to the level of target-cell PD-L1 expression than did the CD28TM. The PD1TM produced an intermediate level of cytokine relative to the CD28TM and TC-210 but rivaled or surpassed the CD28TM when PD-L1 density was high. Moreover, the PD1TM also maintained better viability than the CD28TM when PD-L1 was present at high density, and excessive CD28 stimulation has been reported to induce apoptosis in activated T cell clones (Yu et al. 2003; Yu et al. 2004). In contrast, the CD28TM version of the switch receptor failed to discriminate between low- and-high PD-L1 expression density on target cells and demonstrated an exaggerated response to low levels of PD-L1 in a plate-bound assay. The reduced sensitivity of the CD28TM to PD-L1 levels may be explained by switch receptor homodimerization and/or heterodimerization with endogenous CD28, resulting in amplified signaling (Leddon et al. 2020). For its greater sensitivity to regulation by PD-L1 levels, we selected the PD1TM version of the switch receptor and integrated this with the anti-MSLN TRuC utilized in TC-210 to create a second generation TRuC-T cell product we call TC- 510.

[00587] Expression of the PD1 *CD28 switch receptors did not lead to any significant difference in the surface phenotype of TRuC-T cells when compared to TC-210, except for a slight decrease in the cell surface density of the TRuC receptor. Like TC-210, cytotoxicity, cytokine release, and TRuC activation (pERK) in TC-510 cells is dependent upon TRuC engagement by MSLN. This indicates that the costimulatory activity of the PDlxCD28 switch receptors adheres to the two-step model of T cell activation, which can be an important feature in the context of off tumor toxicity risk.

[00588] The PDlxCD28 switch receptor contained in TC-510 can offer two non-mutually exclusive mechanisms for enhancing TRuC-T cell function: 1.) acting as a PD-1 dominant negative receptor (DNR) that outcompetes endogenous PD-1 for PD-L1 binding, and 2.) delivering a costimulatory signal upon PD-L1 engagement. To elucidate the relative contributions of these two mechanisms, TC-510 and T cells bearing the same TRuC but with the PD1TM switch receptor replaced by either a truncated PD-1 lacking an intracellular signaling domain (PD-l ^Trunc ), or a PD1TM switch receptor in which CD28 signaling was mutationally inactivated (PDlTM ^Mutant ) were compared. TRuC-T cells coexpressing either of these constructs failed to show the heightened cytokine response or ability to boost in vitro expansi on/persistence that are associated with TC-510; rather, they performed comparably to TC-210. These findings support the assertion that CD28 costimulatory signaling is the primary mechanism by which the PD1TM switch receptor enhances TC-510 effector function. The lack of any discernible functional enhancement of TRuC-T cells by the PD-1 DNR constructs tested in these studies is surprising, as others have reported an enhancement of CAR-T cell function by a PD-1 DNR (Cherkassky et al. 2016).

[00589] The observation of enhanced expansion and persistence of TC-510 in the face of serial tumor rechallenge in vitro was further supported by an in vivo study in which TC-510 showed a superior ability to durably protect mice from tumor rechallenge compared to TC-210. This result indicates that integrating a PDlxCD28 switch receptor into TRuC-T cells can enhance their capacity for long-term functional persistence in vivo, which can translate into improved clinical efficacy in human cancer patients with solid tumors.

Example 5: Phase l/II Clinical Trial of TC-510

[00590] A phase 1/2 open-label study to evaluate the safety and efficacy of autologous genetically engineered TC-510 T cells in patients with MSLN-expressing cancers, similar to the study described in Example 2, is conducted.

Phase 1 Objectives

[00591] Primary

• To evaluate the safety of autologous genetically modified TC-510 in patients with MSLN- expressing metastatic or unresectable solid tumors.

• Establish the recommended phase 2 dose (RP2D) according to dose-limiting toxicity (DLT) of defined adverse events.

[00592] Secondary

• To determine the overall response rate (ORR) (complete response [CR] + partial response [PR]) according to Response Evaluation Criteria in Solid Tumors (RECIST) v 1.1 and duration of response (DoR).

• To determine the disease control rate (DCR), defined as a composite of ORR and stable disease (SD) lasting at least 8 weeks.

• To evaluate the survival benefit of autologous genetically modified TC-510 as assessed by progression-free survival (PFS) and overall survival (OS)

• To assess humoral immunogenicity to TC-510

Phase 2 Objectives

[00593] Primary

• To evaluate the efficacy of autologous genetically modified TC-510 in patients with MSLN- expressing metastatic or unresectable solid tumors.

• Endpoints: ORR (CR + PR) and DCR (ORR + SD > 8 weeks) according to RECIST v 1.1. [00594] Secondary

• To evaluate the efficacy of autologous genetically modified TC-510 in patients with MSLN- expressing unresectable, metastatic, or recurrent cancers as assessed by TTR, DoR, PFS, and OS.

• To further evaluate the safety of autologous genetically modified TC-510.

• To assess whether patients experience a response upon subsequent TC-510 infusion(s) Exploratory Objectives (Phase 1 and 2)

• To evaluate TC-510 expansion, persistence, and cytokine production in vivo.

• To evaluate key TC-510 product attributes pre- and post-infusion (e.g., phenotype, functionality).

• To evaluate TC-510 infiltration in tumor tissue.

• To evaluate the correlation of metabolic activity of target and non-target lesions post TC-510 infusion and clinical activity.

• To evaluate immune markers in the tumor microenvironment (e.g., PD-L1 expression) before and after TC-510 infusion and their correlation with toxicity and response.

• To evaluate changes in health-related quality of life following treatment with TC-510 (only Phase 2)

• To evaluate the response to TC-510 by iRECIST.

Study Design

[00595] This first-in-human clinical trial is a phase 1/2 open-label study to evaluate the safety and efficacy of autologous genetically engineered TC-510 in patients with advanced MSLN-expressing cancers. Patients will be screened for leukapheresis eligibility, and if deemed eligible, will undergo a large-volume leukapheresis at the enrolling institution to obtain cells for the manufacture of autologous TC-510. Patients’ peripheral blood mononuclear cells will be processed at the enrolling institution and frozen leukocytes will then be shipped to a central site for further processing. Then, the TC-510 (transduced T cells) will be formulated, cryopreserved, and shipped back to the treating institution. If patients then meet treatment eligibility criteria, they will proceed to receive lymphodepleting chemotherapy with cyclophosphamide and fludarabine followed by TC-510 infusion.

[00596] The study will proceed in 2 sequential phases. The phase 1 portion of the study will evaluate varying doses of TC-510 preceded by lymphodepleting chemotherapy. The lymphodepleting chemotherapy regimen will consist of fludarabine for 4 days (day -7 to day -4) and cyclophosphamide for 3 days (day -6 through day -4).

[00597] It is estimated that 25 to 30 patients will be treated during the dose-escalation phase. A standard 3 + 3 dose escalation strategy will be used to identify the RP2D. [00598] For the phase 1 portion of the study, TC-510 may be administered via a fractionated regimen at any point in the study if deemed appropriate for safety or efficacy reasons. The TC-510 dose may be fractionated such that one-third (approximately 33%) of the TC-510 dose will be administered on day 0 and, if well tolerated, the remaining two-thirds (approximately 67%) of the dose will be administered 7 to 10 days later (i.e., Day 7 - Day 10). Alternatively, the TC-510 dose may be fractionated such that one-half (approximately 50%) of the TC 510 dose will be administered on day 0 and, if well tolerated, the remaining half (approximately 50%) of the dose will be administered 7 to 10 days later (i.e., Day 7 - Day 10). In the event the initial (one-third or one-half depending on the fractionation regimen) dose elicits > grade 3 CRS and/or > grade 2 neurotoxicity, the infusion of the second dose can be delayed until the CRS and/or neurotoxicity regresses to grade 1, or otherwise until treatment is deemed safe by both the treating physician and the Medical Monitor. A delay of the second infusion by more than 7 days from the planned second infusion date must be approved. In the event of significant toxicity observed after the first infusion of TC-510, the Medical Monitor and the Investigator, may determine that the cell dose should be fractionated such that one-third (approximately 33%) of the dose will be administered on day 0 and, if well tolerated, a second-third (approximately 33%) will be administered 7 to 10 days later, and if that fraction is well tolerated, the third and final fraction (approximately 33%) will be administered 7 to 10 days after the second fraction was received. The same delay or hold rules as described above will apply to this fractionation method. In the phase 2 portion of the study, TC-510 will be administered at the RP2D.

[00599] Patients receiving TC-510 in the dose escalation phase must have one of the following cancer diagnoses: MPM, Serous Ovarian Adenocarcinoma, Pancreatic Adenocarcinoma, Colorectal Cancer, triple negative breast cancer (TNBC), NSCLC, or Cholangiocarcinoma. This dose escalation phase will proceed with varying cell doses to determine the RP2D: (50 x 10 ⁶ , 100 x 10 ⁶ , 130 x 10 ⁶ , 160 x 10 ⁶ , and 200 x io ⁶ ). All doses mentioned throughout the protocol denote transduced TC-510. A variation on the target dose of 15% (i.e., ± 15%) will be allowed at each dose level. Each patient enrolled to the dose-escalation phase of the study will receive TC-510 as either a single infusion or in a fractionated regimen.

[00600] Prior to escalation to the next dose level, the safety review team (SRT), comprised of the Study Sponsor Medical Monitor and at least 2 Study Investigators, will review the safety data and make recommendations on further study conduct of phase 1.

[00601] In the event the RP2D is determined for 1 or more specific indications, dose escalation may continue to determine the indication-specific RP2D for other indications where TC-510 may be tolerated at higher doses. Once the RP2D for a specific indication is determined, enrollment of the Phase 2 specific arm for such indication may commence. Protocol stagger, safety observation and dose escalation rules will continue to apply to patients within the indication-specific dose escalation cohort(s) until the indication-specific RP2D is determined.

[00602] The SRT may determine that the presence of PD-L1 expression on a baseline biopsy may be associated with a different toxicity profile after TC-510 infusion compared to that observed in patients with no or negligible PD-L1 levels at baseline. In that case, the SRT may rule to proceed with dose escalation in 2 separate cohorts where patients will be stratified according to baseline PD-L1 levels.

[00603] The Phase 2 portion of the study will evaluate the preliminary antitumor activity (efficacy) and better characterize safety of TC-510 at the selected RP2D. Patients will receive TC-510 at the RP2D and will be enrolled according to their cancer diagnosis to 7 distinct cohorts of up to 20 patients each: MPM, Serous Ovarian Adenocarcinoma, Pancreatic Adenocarcinoma, Colorectal Cancer, Triple Negative Breast Cancer, Non-Small Cell Lung Cancer, or Cholangiocarcinoma. Overall, the phase 2 portion of the study will treat 140 patients.

[00604] Phase 1:

• The key objective of the Phase 1 portion of the study will be the determination of the RP2D based on the observed toxicity profile upon TC-510 infusion and the evaluation of DLTs.

• Five TC-510 dose levels (50 x io ⁶ , 100 x io ⁶ , 130 x io ⁶ , 160 x io ⁶ , and 200 x 10 ⁶ ) will be tested following a lymphodepleting chemotherapy regimen. If excessive toxicity is observed at DL0, de-escalation may occur to DL-1.

• TC-510 may be administered via a fractionated regimen at any point in the study if deemed appropriate for safety or efficacy reasoning by either the safety review team (SRT) or the Medical Monitor.

• Dose escalation will proceed according to a modified 3+3 design.

• A planned stagger of 14 days will be instituted between the first patient infused at each dose level and the 2nd patient.

• Patients 2 and 3 in any given cohort may be infused simultaneously.

• Once the RP2D is identified, up to 10 additional patients may be treated at the RP2D in an expansion cohort to further delineate the toxicity profile of TC-510 prior to advancing to the Phase 2 portion of the study. TC-510 retreatment may be possible for patients in the expansion cohort at the RP2D.

• Approximately 25-30 patients will be treated in the Phase 1 portion of the study.

[00605] Phase 2:

• The objective of this phase will be to further characterize the safety profile of TC-510 and to properly evaluate its efficacy. • Patients will receive TC-510 at the RP2D and will be stratified according to their cancer diagnosis in 7 cohorts: malignant pleural/peritoneal mesothelioma (MPM), serous ovarian adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, triple negative breast cancer, nonsmall cell lung cancer (NSCLC), and cholangiocarcinoma.

• A total of 20 patients will be treated with single agent TC-510 in each one of those cohorts. Up to approximately 140 patients will be treated in the phase 2 portion of the study.

• Patients achieving a best response of SD by 8 weeks post-infusion and those with progression after an initial repones to TC-510 infusion will be allowed to be considered for TC-510 retreatment at the RP2D following a lymphodepleting chemotherapy regimen provided they re-meet the treatment eligibility criteria.

[00606] If the number of TC-510 transduced T cells is less than the minimum dose allowed at any given level, manufacturing of additional transduced T cells from excess banked leukapheresis product will be undertaken to achieve a total dose that meets the target dose requirement. In the event that no banked leukapheresis product is available, a second leukapheresis may be performed. If, despite the latter, a TC-510 dose fails to meet the minimum dose requirement, patients may still be eligible to receive TC-510 and participate in this trial. These patients will be evaluated in a separate patient cohort. However, an additional patient whose TC-510 dose meets the minimum cell dose requirement will be added to the cohort.

Leukapheresis Inclusion/Exclusion Criteria

[00607] Patients will be assessed for and must meet leukapheresis eligibility criteria for leukapheresis at the Leukapheresis Eligibility visit.

[00608] Leukapheresis Inclusion Criteria. A patient must meet the following inclusion criteria to be eligible to undergo leukapheresis:

1. Patient (or legally authorized representative) has voluntarily agreed to participate by giving written informed consent in accordance with International Council on Harmonisation (ICH) Good Clinical Practice (GCP) guidelines and applicable local regulations.

2. Patient is > 18 years of age at the time the Informed Consent is signed.

3. Patient has a pathologically confirmed diagnosis of either epithelioid MPM, Serous Ovarian Adenocarcinoma (Serous Ovarian, Fallopian Tube or Primary Peritoneal cancers will be permitted), Pancreatic Adenocarcinoma, Colorectal Cancer, Triple Negative Breast Cancer, NSCLC, or Cholangiocarcinoma

- For patients with MPM, a pathology report documenting epithelioid histology should be provided. In the event epithelioid histology cannot be clearly confirmed via pathology report, the patient may proceed after discussion with the medical monitor or after confirming adequate MSLN expression

- For patients with triple negative breast cancer, treatment sites must provide proof of ER-negative and PR-negative status (in both cases defined as < 1%). Her2-negative status is defined as 0, 1+, or 2+ by immunohistochemistry (IHC). If IHC 2+, a negative in situ hybridization (FISH, CISH, or SISH) test is required by local laboratory testing.

4. For patients with epithelioid MPM, confirmation of MSLN expression is not required prior to enrollment. For Ovarian, Pancreatic, Colorectal, TNBC, NSCLC, and Cholangiocarcinoma only: Patient’s tumor expresses MSLN on > 50% of tumor cells with 1+, 2+, and/or 3+ intensity by immunohistochemistry at a TCR2 Therapeutics designated central laboratory.

- A banked tumor biopsy is allowed at pre-screening if obtained within 5 years of prescreening consent, otherwise a fresh tumor biopsy will be obtained.

5. Patient has advanced (e.g., metastatic or unresectable) cancer. Unresectable refers to a tumor lesion in which clear surgical excision margins cannot be obtained without leading to significant functional compromise.

6. Prior to TC-510 infusion, patients with MPM, Serous Ovarian Adenocarcinoma, TNBC, Colorectal, and NSCLC must have received at least 1 but no more than 5 systemic therapies for metastatic or unresectable disease. Patients with Pancreatic and Cholangiocarcinoma who are treatment naive may be eligible for TC-510 therapy if they have elected not to pursue front-line therapy. Regardless of tumor type, patients must not exceed 5 prior lines of therapy (excluding bridging therapy and surgical procedures) or except where otherwise specified below:

• MPM

• Patients must have received standard frontline therapy (e.g., platinum -based chemotherapy or checkpoint inhibitor therapy).

• Pancreatic Adenocarcinoma

• Patients must have received frontline therapy with fluorouracil-based (e.g., FOLFIRINOX) or gemcitabine-based (e.g., gemcitabine ± Nab-paclitaxel) regimens for advanced disease or have elected not to pursue front-line therapy

Serous Ovarian Adenocarcinoma

• Patients must have received a platinum-based regimen. • Or if they are carriers of a BRCA1/2 mutation they must have received at least a PARP inhibitor unless the treating physician determines that it is in the patient’s best interest to enroll in the TC-510 trial, deems the patient unsuitable, or the patient elects not to receive PARP inhibitor therapy at that time. PARP inhibitors may be given as maintenance therapy with/without bevacizumab following a prior chemotherapy regimen and be considered a single line of therapy. To be considered maintenance therapy, the interval between prior chemotherapy completion and the initiation of the PARP inhibitor with/without bevacizumab must be less than 8 weeks with no evidence of disease progression during that interval.

Triple Negative Breast Cancer (TNBC)

• Must have received at least 1 prior systemic anti-cancer therapy, including a PARP inhibitor for advanced TNBC with a germline mutation in BRCA1/2, and the combination of pembrolizumab with chemotherapy for advanced TNBC with combined positive score (CPS) > 10.

Colorectal Cancer

• Must have progressed after at least 1 prior standard systemic anti-cancer therapy (e.g., oxaliplatin or irinotecan-based regimens ± bevacizumab), including cetuximab or panitumumab in patients with wild type KRAS or immune checkpoint inhibitors in patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) colorectal cancer.

NSCLC

• Patients must have pathologically confirmed (by histology) diagnosis of NSCLC, which is currently stage 3B or stage 4 disease.

• A patient with non-squamous NSCLC must have been tested for relevant EGFR mutations, ALK translocation or other actionable genomic aberrations (e.g., ROS rearrangement, BRAF V600E mutation) for which FDA-approved targeted therapy is available and, if positive, the patient should have received at least 1 such therapy prior to study enrollment.

• Patients with the EGFR T790M mutation must have received the FDA-approved tyrosine kinase inhibitor osimertinib.

• For patients without an actionable mutation, the patient must have received a currently approved frontline regimen (e.g., immune checkpoint inhibitor-based therapy). G. Cholangiocarcinoma

• Patients must have received at least 1 standard systemic regimen for unresectable or metastatic disease (e.g., gemcitab ine-or 5-FU-containing regimens) or they must have elected not to pursue frontline standard of care therapy.

7. Patient has an Eastern Cooperative Oncology Group performance status 0 or 1.

8. Subject is fit for leukapheresis and has adequate venous access for the cell collection.

9. Subjects must have an absolute lymphocyte count (ALC) >350/pL prior to leukapheresis. Patients with an ALC < 350/pL can still be eligible to proceed with leukapheresis if they have a CD3 count of >150/pL. If the CD3 is <150/pL consultation with the Medical Monitor is required.

[00609] Leukapheresis Exclusion Criteria'. A patient meeting any of the following exclusion criteria is not eligible to undergo leukapheresis:

1. Inability to follow the procedures of the study (e.g., due to language problems, psychological disorders, dementia, confusional state).

2. Patient has received or plans to receive the following therapy/treatment prior to leukapheresis:

• Cytotoxic chemotherapy within 3 weeks of leukapheresis

• Corticosteroids: therapeutic doses of steroids must be stopped > 72 hours prior to leukapheresis. Use of inhaled steroids or topical cutaneous steroids is not exclusionary. Corticosteroid therapy at a pharmacologic dose (> 5 mg/day of prednisone or equivalent doses of other corticosteroids) and other immunosuppressive drugs should be avoided until 3 months after TC-510 T cell administration, unless medically indicated to treat new toxicity. Physiological replacement doses of steroids (up to 5 mg/day of prednisone equivalent, or higher if warranted by the patient’s BMI) may be allowed.

• Immunosuppression: any other immunosuppressive medication must be stopped > 4 weeks prior to leukapheresis, including calcineurin inhibitors, methotrexate or other chemotherapy drugs, mycophenolate, steroids (see above), rapamycin, thalidomide, or immunosuppressive antibodies such as rituximab, anti-tumor necrosis factor, anti-interleukin (IL) 6 or anti-IL6R.

• Use of an anti-cancer vaccine within 2 months in the absence of tumor response. The patient should be excluded if their disease is responding to an experimental vaccine given within 6 months;

• Any previous gene therapy using an integrating vector; • Tyrosine kinase inhibitor (e.g., EGFR inhibitors), PARP inhibitors (e.g., olaparib, rucaparib, niraparib), or KRAS G12C inhibitors (e.g., sotorasib, adagrasib) within 72 hours;

• Any previous allogeneic hematopoietic stem cell transplant;

• Investigational treatment or clinical trial within 4 weeks or 5 half-lives of investigational product, whichever is shorter.

• Coronavirus disease 2019 (Covid- 19; SARS-CoV-2) vaccine dose within 4 weeks from estimated date of leukapheresis, unless approved by TCR2 Therapeutics Medical Monitor. CNS disease/brain metastases: a. Patients with leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases: patients are eligible if they have completed their treatment, have recovered from the acute effects of radiation therapy or surgery prior to study entry, and a) have no evidence of brain metastases post treatment or b) are asymptomatic, have discontinued corticosteroid treatment or anti-seizure medications for these metastases for at least4 weeks and have radiographically stable CNS metastases without associated edema or shift for at least 3 months prior to study entry (note: prophylactic anti-seizure medications are acceptable; up to 5 mg per day of prednisone or equivalent will be allowed, or higher if warranted by the patient’s BMI). Patient has any other prior or concurrent malignancy with the following exceptions:

• Adequately treated basal cell or squamous cell carcinoma (adequate wound healing is required prior to study entry).

• In situ carcinoma of the cervix or breast, treated curatively and without evidence of recurrence for at least 12 months prior to the study.

• Treated non-melanoma skin cancer.

• Stage 0 or 1 melanoma completely resected at least 12 months prior to the study.

• Successfully treated organ-confined prostate cancer with no evidence of progressive disease based on PSA levels and are not on active therapy.

• A primary malignancy which has been completely resected and in complete remission for 5 years.

• Other malignancies deemed unlikely to be of clinical significance during TC-510 therapy. 5. Patient has active infection with HIV, hepatitis B virus, HCV, or HTLV as defined below:

• Positive serology for HIV, HTLV-1, or HTLV-2.

• Active hepatitis B infection as demonstrated by test for hepatitis B surface antigen. Patients who are hepatitis B surface antigen negative but are hepatitis B core antibody positive must have undetectable hepatitis B DNA and receive prophylaxis against viral reactivation.

• Active hepatitis C infection as demonstrated by hepatitis C RNA test. Patients who are HCV antibody positive will be screened for HCV RNA by any reverse transcription PCR or branched DNA assay. If HCV antibody is positive, eligibility will be determined based on a negative screening RNA value.

6. Patient with pulse oximetry < 90% on room air or has one of the following will be excluded

• Radiographic evidence of underlying interstitial lung disease.

• Active interstitial lung disease/pneumonitis or a history of interstitial lung disease/pneumonitis requiring therapy with systemic corticosteroids

7. History of autoimmune or immune mediated disease such as multiple sclerosis, lupus, rheumatoid arthritis, inflammatory bowel disease, or small vessel vasculitis.

Treatment Inclusion/Exclusion Criteria

[00610] A patient must meet the following treatment inclusion criteria to be eligible to proceed with first protocol defined therapy (e.g., Lymphodepletion). Treatment Eligibility will be formally assessed at Baseline. For retreatment with TC-510, the following criteria also apply.

[00611] Treatment Inclusion Criteria: A patient must meet the following inclusion criteria to be eligible to receive therapy on this study:

• Patient (or legally authorized representative) has voluntarily agreed to participate by giving written informed consent in accordance with International Conference on Harmonisation Good Clinical Practice guidelines and applicable local regulations.

• Patient has agreed to abide by all protocol required procedures including study -related assessments, and management by the treating institution for the duration of the study and LTFU.

• Patient is > 18 years of age at the time the Informed Consent is signed.

• Patient has a pathologically confirmed diagnosis of either MPM, Serous Ovarian Adenocarcinoma (patients with Serous Ovarian, Fallopian Tube or Primary Peritoneal cancers will be permitted), Pancreatic Adenocarcinoma, Colorectal Cancer, TNBC, NSCLC, or Cholangiocarcinoma. • For patients with MPM, a pathology report documenting epithelioid histology should be provided. In the event epithelioid histology cannot be clearly confirmed via pathology report, the patient may proceed after discussion with the medical monitor or confirming adequate MSLN expression.

• For patients with TNBC, treatment sites must provide proof of ER-negative and PR-negative status (in both cases defined as < 1%). Her2-negative status is defined as 0, 1+, or 2+ by IHC. If IHC 2+, a negative in situ hybridization (FISH, CISH, or SISH) test is required by local laboratory testing.

• For patients with epithelioid MPM, confirmation of MSLN expression is not required prior to treatment. For Ovarian, Pancreatic, Colorectal, TNBC, NSCLC, and Cholangiocarcinoma only: Patient’s tumor expresses MSLN on > 50% of tumor cells with 1+, 2+, and/or 3+ intensity by immunohistochemistry at a designated central laboratory:

• For all patients, MSLN testing must be performed on a biopsy sample obtained within 6 months of the planned first protocol defined therapy. The pre-screening biopsy may fulfill the Baseline biopsy requirement if collected within 6 months of the start of treatment (provided there is adequate tissue for correlative testing). Otherwise, a new biopsy sample from within 6 months of the first protocol defined therapy should be submitted for MSLN testing. If deemed unsafe by the principal investigator and/or the lesion is inaccessible, the patient may be allowed to proceed without the Baseline biopsy.

• Patient has advanced (metastatic or unresectable) cancer. Unresectable refers to a tumor lesion in which clear surgical excision margins cannot be obtained without leading to significant functional compromise.

• Patient has at least 1 lesion that meets evaluable and measurable criteria confirmed RECIST v 1.1. Patients who have received prior local therapy (including but not limited to embolization, chemoembolization, radiofrequency ablation, or radiation therapy) are eligible provided measurable disease falls outside of the treatment field or within the field and has shown >20% growth in size since post-treatment assessment.

• The patient must not have required a paracentesis or thoracentesis within the preceding 4 weeks of TC-510 infusion nor be projected to require a paracentesis or thoracentesis within the next 8 weeks. Patients with catheters in place for frequent drainage may be allowed.

• Prior to TC-510 infusion, patients with MPM, Serous Ovarian Adenocarcinoma, TNBC, Colorectal, and NSCLC must have received at least 1 but no more than 5 systemic therapies for metastatic or unresectable disease. Patients with Pancreatic and Cholangiocarcinoma who are treatment naive may be eligible for TC-510 therapy if they have elected not to pursue front-line therapy. Regardless of tumor type, patients must not exceed 5 prior lines of therapy (excluding bridging therapy and surgical procedures) or except where otherwise specified below:

• MPM: Patients must have received standard frontline therapy (e.g., platinum-based chemotherapy or checkpoint inhibitor therapy).

• Pancreatic Adenocarcinoma: Patients must have received frontline therapy with fluorouracilbased (e.g., FOLFIRINOX) or gemcitabine-based (e.g., gemcitabine ± Nab-paclitaxel) regimens for advanced disease or have elected not to pursue standard front-line therapy.

• Serous Ovarian Adenocarcinoma: Patients must have received a platinum-based regimen.

• Or if they are carriers of a BRCA1/2 mutation they must have received at least a PARP inhibitor unless the treating physician determines that it is in the patient’s best interest to enroll in the TC-510 trial, deems the patient unsuitable, or the patient elects not to receive PARP inhibitor therapy at that time. PARP inhibitors may be given as maintenance therapy with/without bevacizumab following a prior chemotherapy regimen and be considered a single line of therapy. To be considered maintenance therapy, the interval between prior chemotherapy completion and the initiation of the PARP inhibitor with/without bevacizumab must be less than 8 weeks with no evidence of disease progression during that interval.

• Triple Negative Breast Cancer: Must have received at least 1 prior systemic anti-cancer therapy, including a PARP inhibitor for advanced TNBC with a germline mutation in BRCA1/2, and the combination of pembrolizumab with chemotherapy for advanced TNBC with combined positive score (CPS) > 10.

• Colorectal Cancer: Must have progressed after at least 1 prior standard systemic anti-cancer therapy (e.g., oxaliplatin or irinotecan-based regimens ± bevacizumab), including cetuximab or panitumumab in patients with wild type KRAS or immune checkpoint inhibitors in patients with MSI-H or dMMR colorectal cancer.

• NSCLC: i) Patients must have pathologically confirmed (by histology) diagnosis of NSCLC which is currently stage 3B or stage 4 disease. ii) A patient with non-squamous NSCLC must have been tested for relevant EGFR mutations, ALK translocation or other actionable genomic aberrations (e.g., ROS rearrangement, BRAF V600E mutation) for which FDA-approved targeted therapy is available and, if positive, the patient should have received at least 1 such therapy prior to study enrollment. iii) Patients with the EGFR T790M mutation must have received the FDA-approved tyrosine kinase inhibitor osimertinib.

• Cholangiocarcinoma: i) Patients must have received at least 1 standard systemic regimen for unresectable or metastatic disease (e.g., gemcitabine-or 5-FU-containing regimens) or they must have elected not to pursue frontline standard of care therapy. ii) Patients must have measurable disease, defined as at least 1 lesion that can be accurately measured in at least 1 dimension (longest diameter to be recorded for non-modal lesions and short axis for nodal lesions) as > 20 mm (>2 cm) with conventional techniques or as > 10mm (>lcm) with computed tomography (CT) scan or magnetic resonance imaging (MRI).

• Patient has an Eastern Cooperative Oncology Group performance status 0 or 1.

• All patients must have undergone a rapid influenza diagnostic test and/or a respiratory viral panel (including coronavirus disease 2019 (Covid-19; SARS-CoV-2) as per Institutional guidelines within 14 days prior to the first protocol defined therapy. If the patient is positive for influenza, oseltamivir phosphate or zanamivir should be administered for 10 days (see Tamiflu® or Relenza® package insert for dosing). The patient must complete their 10-day treatment course prior to receiving TC-510.

• For patients residing in the US, Canada, Europe and Japan, influenza testing is required during the months of October through May (inclusive).

• For patients residing in the southern hemisphere such as Australia, influenza testing is required during the months of April through November (inclusive).

• For patients with significant international travel, both calendar intervals above may need to be considered.

• In the event a patient tests positive for coronavirus disease 2019 (Covid- 19; SARS-CoV-2), TC-510 infusion should be delayed until the patient is asymptomatic and deemed fit for infusion by the treating physician.

• Patient has a left ventricular ejection fraction > 45% as measured by resting echocar di ogram/MUGA, with no clinically significant pericardial effusion.

• FPCP must have a negative urine or serum pregnancy test (FPCP is defined as premenopausal and not surgically sterilized). FPCP must agree to use effective birth control or to abstain from heterosexual activity throughout the study, starting on the day of first dose of lymphodepl eting chemotherapy through 12 months post TC-510 infusion. Effective contraceptive methods include intra-uterine device, oral or injectable hormonal contraception, or 2 adequate barrier methods (e.g., diaphragm with spermicide, cervical cap with spermicide, or female condom with spermicide). Spermicides alone are not an adequate method of contraception.

• Or: Male patients must be surgically sterile or agree to use a double barrier contraception method or abstain from heterosexual activity with a female of childbearing potential starting at the first dose of protocol -defined treatment and for 4 months thereafter or longer (if indicated in the country specific monograph/label for cyclophosphamide).

• Patient must have adequate organ function as indicated by the laboratory values in Table 3 above. [00612] Treatment Exclusion Criteria'. A patient meeting any of the following exclusion criteria is not eligible for participation in the treatment portion of this study:

• Inability to follow the procedures of the study (e.g., due to language problems, psychological disorders, dementia, confusional state).

• Known or suspected non-compliance, drug, or alcohol abuse.

• Participation in another study with investigational drug within the 28 days or 5 half-lives of the drug, whichever is shorter, preceding and during the present study.

• Patient is pregnant (or intends to become pregnant during the course of the study) or breastfeeding.

• Patient has received or plans to receive the following therapy/treatment prior to the first protocol- defined therapy:

• Cytotoxic chemotherapy within 3 weeks of TC-510 T cell infusion.

• Corticosteroids: therapeutic doses of steroids must be stopped at least 2 weeks prior to TC-510 T cell infusion. Use of inhaled steroids or topical cutaneous steroids is not exclusionary. Corticosteroid therapy at a pharmacologic dose (> 5 mg/day of prednisone or equivalent doses of other corticosteroids) and other immunosuppressive drugs should be avoided until 3 months after TC-510 T cell administration, unless medically indicated to treat new toxicity. Physiological replacement doses of steroids (up to 5 mg/day of prednisone equivalent, or higher if warranted by the patient’s BMI) may be allowed.

• Any other immunosuppressive medication must be stopped > 4 weeks prior to first protocol defined treatment, including (but not limited to) calcineurin inhibitors, methotrexate or other chemotherapy drugs, mycophenolate, steroids (see above), rapamycin, thalidomide, or immunosuppressive antibodies such as rituximab, anti-tumor necrosis factor, anti-interleukin (IL) 6 or anti-IL6R.

• Anti-PD-1 monoclonal antibody therapy must be stopped > 4 weeks prior to first protocol- defined treatment. • Use of an anti-cancer vaccine within 2 months in the absence of tumor response. The patient should be excluded if their disease is responding to an experimental vaccine given within 6 months;

• Any previous gene therapy using an integrating vector (except for TC-510 in the case of retreatment);

• Small molecule tyrosine kinase inhibitors (e.g., EGFR inhibitors), PARP inhibitors (e.g., olaparib, rucaparib, niraparib), or KRAS G12C inhibitors (e.g., sotorasib, adagrasib) within 72 hours;

• Any previous allogeneic hematopoietic stem cell transplant;

• Investigational treatment or clinical trial within 4 weeks or 5 half-lives of investigational product, whichever is shorter;

• Radiotherapy to the target lesions within 4 weeks prior to lymphodepleting chemotherapy. A lesion with unequivocal progression may be considered a target lesion regardless of time from last radiotherapy dose. NOTE: There is no washout period for palliative radiation to non-target lesions;

• Hepatic radiation, chemoembolization, and/or radiofrequency ablation within 4 weeks.

• Immune therapy (e.g. monoclonal antibody therapy, checkpoint inhibitors) within 4 weeks.

• Toxicity from previous anti-cancer therapy that has not recovered to < grade 1 (except for nonclinically significant toxicities, e.g., alopecia, vitiligo). Patients with grade 2 toxicities that are deemed stable or irreversible (e.g., peripheral neuropathy) may be eligible.

• History of allergic reactions attributed to compounds of similar chemical or biologic composition to fludarabine, cyclophosphamide, or other agents used in the study.

• History of autoimmune or immune mediated disease such as multiple sclerosis, lupus, rheumatoid arthritis, inflammatory bowel disease, or small vessel vasculitis.

• Maj or surgery (other than diagnostic surgery) within 4 weeks prior to first protocol defined therapy, minor surgery including diagnostic surgery within 2 weeks (14 days) excluding central intravenous port placements and needle aspirate/core biopsies. Radio frequency ablation or transcatheter arterial chemoembolization within 6 weeks prior to first protocol defined therapy.

• Central nervous system (CNS) disease/brain metastases:

• Patients with leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases: patients are eligible if they have completed their treatment, have recovered from the acute effects of radiation therapy or surgery prior to study entry, and a) have no evidence of brain metastases post treatment or b) are asymptomatic, have discontinued corticosteroid treatment or anti-seizure medications for these metastases for at least 4 weeks and have radiographically stable CNS metastases without associated edema or shift for at least 3 months prior to study entry (note: prophylactic anti-seizure medications are acceptable; up to 5 mg/day of prednisone or equivalent will be allowed; higher doses may be allowed if warranted due to patient BMI).

• Patient has any other prior or concurrent malignancy with the following exceptions:

• Adequately treated basal cell or squamous cell carcinoma (adequate wound healing is required prior to study entry)

• In situ carcinoma of the cervix or breast, treated curatively and without evidence of recurrence for at least 12 months prior to enrollment

• Treated non-melanoma skin cancer

• Stage 0 or 1 melanoma completely resected at least 12 months prior to enrollment

• Successfully treated organ-confined prostate cancer with no evidence of progressive disease based on prostate specific antigen (PSA) levels and are not on active therapy

• A primary malignancy which has been completely resected and in complete remission for > 5 years

• Malignancies deemed unlikely to be of clinical significance during TC-510 T cell therapy

• Patient has an electrocardiogram showing a clinically significant abnormality at screening or showing an average QTc interval > 450 msec in males and > 470 msec in females (> 480 msec for patients with bundle branch block). Either Fridericia’s or Bazett’s formula may be used to correct the QT interval.

• Patient has uncontrolled intercurrent illness including, but not limited to:

• Ongoing or active infection; e.g., sepsis, prolonged unresolved bacterial cholangitis with destruction of bile duct branches (e.g., after endoprosthesis insertion) or 2 or more cholangitis in the last 6 months.

• Clinically significant cardiac disease defined by congestive heart failure New York Heart Association class 3 or class 4;

• Uncontrolled clinically significant arrhythmia;

• Acute coronary syndrome (angina or myocardial infarction), stroke, or peripheral vascular event in the last 6 months;

• Serious thrombotic event in the last 6 months. • Interstitial lung disease (patients with existing pneumonitis as a result of radiation are not excluded; however, patients must not be oxygen dependent as demonstrated by oxygen saturation < 90% on room air);

• Liver cirrhosis or primary sclerosing cholangitis.

• Patient has active infection with human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus (HCV), or human T-lymphotropic virus (HTLV) as defined below:

• Positive serology for HIV, HTLV- 1 , or HTL V-2;

• Active hepatitis B infection as demonstrated by test for hepatitis B surface antigen. Patients who are hepatitis B surface antigen negative but are hepatitis B core antibody positive must have undetectable hepatitis B deoxyribonucleic acid (DNA) and receive prophylaxis against viral reactivation;

• Active hepatitis C infection as demonstrated by hepatitis C ribonucleic acid (RNA) test. Patients who are HCV antibody positive will be screened for HCV RNA by any reverse transcription polymerase chain reaction (PCR) or branched DNA assay. If HCV antibody is positive, eligibility will be determined based on a negative screening RNA value.

Study Treatment

[00613] Study Treatment is as above in Example 2, including the lymphocyte depletion treatment schedule set forth in Table 4 (for patients with renal dysfunction as shown in Table 5) and the dose escalation s set forth in Table 2.

[00614] The dose of fludarabine (set forth in Tables 4 and 5) may be rounded down to the closest vial as long as the final fludarabine dose is within 10% of the calculated planned dose (e.g., 30 mg/m ² or 20 mg/m ² for patients with renal dysfunction). Two alternative lymphodepleting chemotherapy regimens exist in the event of fludarabine shortage. a. Alternative 1 : bendamustine 100 mg/m ² /day IV on days -5 and -4 (i.e., 2 doses). b. Alternative 2: cyclophosphamide 600 mg/m ² /day IV on day s-6, -5, and -4 (i.e., 3 doses) and oxaliplatin 130 mg/m ² IV on day -6 (i.e., 1 dose)

[00615] In the event the patient has an unforeseeable delay or missed dosing day for this lymphodepleting regimen, the missed dose will be administered, and the TC-510 dosing day will be moved back, maintaining the last dosing day at -4 relative to TC-510 infusion (or day -5 for retreatment due to the abbreviated lymphodepleting therapy regime described below).

[00616] In Phase 1 and Phase 2, patients receiving TC-510 must have one of the following cancer diagnoses: MPM, serous ovarian adenocarcinoma, pancreatic adenocarcinoma, colorectal cancer, triple negative breast cancer (TNBC), NSCLC, or cholangiocarcinoma. [00617] After the completion of lymphodepleting chemotherapy, the Investigator should ensure that the following safety criteria are met prior to TC-510 infusion:

• The patient completed all days of lymphodepleting chemotherapy

• No significant worsening of any clinical condition since the start of lymphodepleting chemotherapy

• No suspected or active systemic infection

• No onset of fever > 38°C / 100.4°F since the last dose of lymphodepleting chemotherapy

• No requirement for supplemental 02 to keep 02 saturation on room air (SaO2) > 92%

• No presence of progressive radiographic abnormalities (if chest X-ray was performed)

• The Investigator deems that it is safe for the patient to proceed with TC-510 infusion

[00618] If per the assessment of the Investigator, the patient does not meet any of the above criteria, the TC-510 infusion should be held until consultation with the Medical Monitor.

[00619] On day 0 of the study, patients participating in the phase 1 portion of the study will receive TC-510 within the dose range of 50 x 10 ⁶ to 200 x 10 ⁶ transduced cells by IV infusion. The recommended dose for patients participating in the phase 2 portion will be determined at the end of the dose-escalating phase 1.

[00620] A planned stagger of 14 days will be instituted between the first patient infused at each dose level and the 2 ^nd patient.

[00621] TC-510 T Cell Re treatment (Applicable during Phase 1 RP2D Expansion Cohort and Phase 2:

[00622] In the phase 1 RP2D expansion cohort and in the phase 2 portion of the study, patients will be candidates for TC-510 retreatment if they fall into either of the following groups:

• Patients who have an obj ective response (i.e., PR or CR as assessed by the Investigator) after TC-510 infusion and develop signs and symptoms of progression.

• Patients whose best response to a prior TC-510 infusion is SD (as assessed by the Investigator) sustained for at least 8 weeks post-TC-510 infusion

[00623] The decision to undergo subsequent TC-510 regimen will be made by the treating Investigator and after consultation and in alignment with the TCR2 Therapeutics Medical Monitor. TC-510 retreatment will follow similar procedural requirements as the initial dose, including the posttreatment study requirements, unless otherwise specified below:

• Patients will be required to meet the original treatment eligibility criteria again and should not have received any other therapy for their underlying malignancy. • Patients will be required to undergo a biopsy, however, they will not be required to undergo MSLN testing and demonstrate MSLN expression. A post infusion, on-study biopsy (e.g. Week 6) within 3 months prior to the planned retreatment may fulfill the retreatment biopsy requirement (provided there is adequate tissue for correlative testing).

• Retreatment will consist of an abbreviated course of lymphodepleting chemotherapy followed by TC-510 infusion:

• Lymphodepleting chemotherapy will consist of fludarabine 30 mg/m2/day on days -7 through -5 (i.e., 3 doses) and cyclophosphamide 600 mg/m2/day on days -6 through -5 (i.e., 2 doses). There will be no chemotherapy given on day -4. This regimen differs from that given prior to the initial TC-510 infusion.

• In the event of retreatment and if bendamustine must be used, the regimen should be abbreviated to 70 mg/m2/day IV on days -5 and -4 (i.e. 2 doses)

• In the event the patient has an unforeseeable delay or missed dosing day for this lymphodepleting regimen, the missed dose will be administered, and the TC- 510 dosing day will be moved back, maintaining the last dosing day at -5 for retreatment).

[00624] Patients meeting treatment eligibility criteria for a second dose may proceed to TC-510 infusion no sooner than 8 weeks and no later than 1 year (52 weeks) following completion of the first TC-510 dose.

[00625] Patients eligible for retreatment should be reconsented following discussion regarding benefits and risks of TC-510 therapy and if applicable, a careful explanation about the need to undergo leukapheresis a second time for the manufacturing of TC-510 prior to performing any study related procedures or treatment. This conversation should be recorded in the patient’s source document.

[00626] For retreatment (phase 1 RP2D expansion cohort and phase 2), the same infusion process will be followed.

Study Duration and Completion

[00627] For each individual patient, the length of study participation includes a screening period that will account for the time from signing the pre-screening ICF to leukapheresis, a 4-day conditioning chemotherapy treatment period (as applicable), a TC-510 treatment period (which may include an in- hospital period), and a post-treatment assessment period lasting a maximum of 108 weeks, and longterm follow-up for up to approximately 5 years post-infusion. Thus, for patients who complete the entire protocol from the date of pre-screening informed consent through the completion of up to approximately 5 years of long-term follow up, the duration of the study may be approximately 5 years and 2 months. However, individual study duration will vary depending on a patient’s prescreening requirements, potential retreatments (phase 1 RP2D expansion cohort and phase 2 only) and survival.

[00628] Study procedures, correlative studies and research assessments, and safety evaluations are as set forth in Example 2.

Endnotes

[00629] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

APPENDIX A: SEQUENCE SUMMARY

Table 6. Sequences