COMPRISING A STAP DOMAIN AND A KINASE DOMAIN OR A STAP DOMAIN AND A

PHOSPHATASE DOMAIN

TECHNICAL FIELD

This document relates to methods and materials involved in treating cancer. For example, this document provides chimeric antigen receptors (CARs) that include: (a) an intracellular component containing a signal transduction domain (e.g, a kinase domain or a phosphatase domain) and a signal transducing adaptor protein (STAP) domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain. For example, this document provides methods and materials for using T cells presenting CARs (CAR-T cells) provided herein for treating a mammal (e.g, a human) having cancer.

BACKGROUND

CAR-T cells and natural killer cells presenting CARs (CAR-NK cells) have been successful as adoptive cell therapies for the treatment of certain hematological cancers. However, treatment of other types of cancer has been less successful in the clinic. Canonical chimeric antigen receptor (CAR) designs are limited by using the intracellular domains of costimulatory receptors and the T cell receptor (TCR) z-chain. In order to engineer CAR-T cells that have improved treatment efficacy, new CAR designs are needed.

SUMMARY

This document provides methods and materials involved in treating cancer. For example, this document provides chimeric antigen receptors (CARs) that include: (a) an intracellular component containing a signal transduction domain ( e.g ., a kinase domain or a phosphatase domain) and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain as well as CAR-T cells presenting one or more CARs provided herein. For example, this document provides methods and materials for using CAR-T cells presenting one or more CARs provided herein for treating a mammal (e.g., a human) having cancer. In some cases, a mammal (e.g, a human such as a human having cancer) can be administered CAR-T cells presenting one or more CARs provided herein to stimulate a signal transduction pathway (e.g., a signal transduction pathway that involves the signal transduction domain of the CAR) thereby inducing one or more cellular responses.

As described herein, a signal producing T cell stimulator (SPRTS)-CAR that includes ( e.g ., is designed to include) an intracellular component containing a signal transduction domain (e.g., a kinase domain or a phosphatase domain) and a STAP domain and is presented on a T cell (e.g, a SPRTS-CAR-T Cell) can be used to modulate one or more cellular responses by directly activating one or more polypeptide components of a particular signaling pathway. In some cases, cellular responses within the SPRTS-CAR-T Cell can be modulated. In some cases, cellular responses of one or more cells nearby (e.g, in contact with) the SPRTS-CAR-T Cell can be modulated. For example, a SPRTS-CAR described herein can be used to modulate one or more cellular responses upon activation of the SPRTS- CAR by binding between the SPRTS-CAR and an antigen that is recognized by the SPRTS- CAR. As such, SPRTS-CAR-T Cells can be used to modulate specific cellular responses in the presence of specific antigens.

Having the ability to modulate specific cellular responses in the presence of specific antigens using the CARs described herein (e.g, SPRTS-CARs including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) provides a unique and unrealized opportunity to direct a cellular response to particular stimulus (e.g, a particular antigen).

In general, one aspect of this document features CARs including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain. The antigen binding domain can be an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), or a single domain camelid antibody binding domain (VHH). The antigen binding domain can be a scFv domain. The antigen binding domain can target a tumor-specific antigen. The tumor-specific antigen can be a cluster of differentiation 19 (CD 19) antigen, a CD22 antigen, a CD20 antigen, a GD2 antigen, a EGFRvIII antigen, a mesothelin antigen, a IL-13RA antigen, a B-cell maturation antigen (BCMA) antigen, a CD138 antigen, a NKG2-D antigen, a HER2/Neu antigen, a IL-13RA2 antigen, a CD137 antigen, a CD28 antigen, a B7- H3 (CD276) antigen, aCD16V antigen, a CA-125 antigen, a MUC-1 antigen, a mutated p53 antigen, a mutated Ras antigen, a ERBB2 (HER2) antigen, a folate binding protein (FBP) antigen, aB7-H6 antigen, a CD123 antigen, aLlCAM (CD171) antigen, a CD30 antigen, a CD33 antigen, a CD37 antigen, a CD38 antigen, a CD4 antigen, a CD47 antigen, a CD70 antigen, a CD73 antigen, a CD79b antigen, a CLL1 (CD371) antigen, a CEA antigen, a Claudin 18.1 antigen, a Claudin 18.2 antigen, a CS-1 antigen, a CSPG4 antigen, a EFGRvIII antigen, aENPP3 antigen, a EpCAM antigen, a EphA2 antigen, a ErbB antigen, a FAP antigen, a FRa antigen, a GD3 antigen, a Glypican 3 antigen, a IL-llRa antigen, a LewisY/LeY antigen, a MUC-16 antigen, a NGFR antigen, a PDl antigen, aPD-Ll antigen, a PSCA antigen, a PSMA antigen, a ROR1 antigen, a SLAMF7 antigen, a TACI antigen, a TAG72 antigen, a EILBP antigen, a MICA/B antigen, a VEGFR2 antigen, a WT1 antigen, a protein-tyrosine kinase-7 (PTK7) antigen, a six-transmembrane epithelial antigen of the prostate (STEAP) 1 antigen, a STEAP2 antigen, a programmed cell death 1 ligand 2 (PD-L2) antigen, a c-Met antigen, a CD70 antigen, a MG7 antigen, a epidermal growth factor receptor 1 (EGFR1) antigen, a platelet-derived growth factor receptor (PDGFR) 1/2 antigen, a Nectin- 4 antigen, a CD 133 antigen, a DNAX accessory molecule- 1 (DNAM-1) antigen, a fms-like tyrosine kinase 3 (FLT-3) antigen, a ETLBPl-6 antigen, a CD112 antigen, a CD44 antigen, or a TROP2 antigen. The transmembrane domain can be a T cell receptor a chain transmembrane domain, a T cell receptor b chain transmembrane domain, a Eϋ3z chain transmembrane domain, a CD28 transmembrane domain, a CD3e transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD 16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, a CD278 transmembrane domain, a CD 154 transmembrane domain, a CD357 transmembrane domain, a CD2 transmembrane domain, a CD6 transmembrane domain, a CD1 la transmembrane domain, a CD1 lc transmembrane domain, a CD 14 transmembrane domain, a CD 18 transmembrane domain, a CD20 transmembrane domain, a CD25 transmembrane domain, a CD30 transmembrane domain, a CD32 transmembrane domain, a CD34 transmembrane domain, a CD40 transmembrane domain, a CD43 transmembrane domain, a CD47 transmembrane domain, a CD49 transmembrane domain, a CD52 transmembrane domain, a CD56 transmembrane domain, a CD57 transmembrane domain, a CD58 transmembrane domain, a CD69 transmembrane domain, a CD70 transmembrane domain, a CD94 transmembrane domain, a CD95 transmembrane domain, a CD 123 transmembrane domain, a CD 133 transmembrane domain, a CD138 transmembrane domain, a CD 152 transmembrane domain, a CD 159a transmembrane domain, a CD223 transmembrane domain, a CD274 transmembrane domain, a CD276 transmembrane domain, a CD279 transmembrane domain, a CD314 transmembrane domain, a CD366 transmembrane domain, or a CD371 transmembrane domain. The transmembrane domain can be a CD8 transmembrane domain. The STAP domain can be a SH2-domain-containing leukocyte protein of 76 kDa (SLP76) polypeptide STAP domain, a Grb2-family adaptor downstream of She (Gads) polypeptide STAP domain, a linker of activation of T cells (LAT) polypeptide STAP domain, a TNF receptor associated factor (TRAF) 1 polypeptide STAP domain, a TRAF2 polypeptide STAP domain, a TRAF3 polypeptide STAP domain, a TRAF5 polypeptide STAP domain, a growth factor receptor-bound protein 2 (Grb2) polypeptide STAP domain, a son of sevenless (SOS) polypeptide STAP domain, aNF-kappa-B essential modulator (NEMO) polypeptide STAP domain, or a TGF -beta-activated kinase (TAB) 1/2 polypeptide STAP domain. The kinase domain can be a lymphocyte specific protein tyrosine (Lck) kinase domain, a Z chain-associated protein kinase 70 (ZAP-70) kinase domain, an interleukin-2-inducible T cell tyrosine (ITK) kinase domain, a receptor-interacting protein (RIP) kinase domain, an IKB kinase (IKK) kinase domain, an apoptosis signal-regulating kinase 1 (ASK-1) kinase domain, an extracellular signal-regulated kinase (ERK) kinase domain, a F-kappa-B -inducing kinase (NIK) kinase domain, a mitogen-activated protein kinase (MEKK1) kinase domain, a mitogen-activated protein kinase 14 (MAP3K14)) kinase domain, a Src polypeptide kinase domain, a Yes polypeptide kinase domain, a Lyn polypeptide kinase domain, a Fyn polypeptide kinase domain, a Fgr polypeptide kinase domain, a Yrk polypeptide kinase domain, a Bruton's tyrosine kinase (Btk) polypeptide kinase domain, a Bmx polypeptide kinase domain, a Txk polypeptide kinase domain, a Tec polypeptide kinase domain, a spleen tyrosine kinase (Syk) polypeptide kinase domain, a focal adhesion kinase (FAR) polypeptide kinase domain, a Pyk2 polypeptide kinase domain, a C- terminal Src kinase (Csk) polypeptide kinase domain, an epidermal growth factor receptor (EGFR) polypeptide kinase domain, or a platelet-derived growth factor receptor (PDGFR) polypeptide kinase domain. In some implementations the kinase domain can include an amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some implementations the kinase domain can include an amino acid sequence having from one to five (e.g. one, two, three, four, or five) conservative substitutions as compared to an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some cases, the antigen binding domain can be a scFv that can target ROR1, the STAP domain can be a SLP76 STAP domain, and the kinase domain can be a ZAP-70 kinase domain.

In another aspect, this document features T cells comprising one or more CARs including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain.

In another aspect, this document features nucleic acid constructs comprising a nucleic acid sequence encoding a CAR including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain. The nucleic acid sequence encoding the CAR can include a CD8a secretion signal (e.g., a CD8a secretion signal). The nucleic acid construct can include a promoter operably linked to the nucleic acid sequence encoding the CAR. The promoter can be a U6 promoter, a HI promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a b-actin promoter, a phosphoglycerol kinase (PGK) promoter, an MND promoter, an EF la promoter, a MND promoter, or a mCAG promoter. The nucleic acid sequence encoding the CAR can include a polyadenylation signal. In another aspect, this document features T cells including a nucleic acid construct of comprising a nucleic acid sequence encoding a CAR including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain, where the nucleic acid construct expresses the CAR.

In another aspect, this document features pharmaceutical compositions including T cells comprising one or more CARs including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain and/or T cells including a nucleic acid construct of comprising a nucleic acid sequence encoding a CAR including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain, where the nucleic acid construct expresses the CAR, and a pharmaceutically acceptable excipient.

In another aspect, this document features methods for treating a mammal having cancer. The methods can include or consist essentially of administering to a mammal having cancer T cells comprising one or more CARs including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain and/or T cells including a nucleic acid construct of comprising a nucleic acid sequence encoding a CAR including an extracellular component comprising an antigen binding domain; a transmembrane domain; and an intracellular component comprising a kinase domain and a STAP domain, where the nucleic acid construct expresses the CAR; where the CAR targets an antigen expressed by the cancer. The mammal can be a human. The cancer can be prostate cancer, ovarian cancer, cervical cancer, colorectal cancer, intestinal cancer, testicular cancer, skin cancer, lung cancer, thyroid cancer, bone cancer, breast cancer, bladder cancer, uterine cancer, vaginal cancer, pancreatic cancer, liver cancer, kidney cancer, brain cancer, spinal cord cancer, oral cancer, parotid tumor, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, myeloma, soft tissue sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, Ewing sarcoma, osteosarcoma, gastrointestinal stromal tumor (GIST), penile cancer, or cervical cancer. The administering can include intravenous injection.

In another aspect, this document features CARs including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain. The antigen binding domain can be an Fab, a VH chain, a VL chain, a scFv, or a VHH. The antigen binding domain can be a scFv domain. The antigen binding domain can target a tumor-specific antigen. The tumor-specific antigen can be a CD 19 antigen, a CD22 antigen, a CD20 antigen, a GD2 antigen, a EGFRvIII antigen, a mesothelin antigen, a IL-13RA antigen, a BCMA antigen, a CD138 antigen, a NKG2-D antigen, a HER2/Neu antigen, a IL-13RA2 antigen, a CD137 antigen, a CD28 antigen, a B7-H3 (CD276) antigen, aCD16V antigen, a CA-125 antigen, a MUC-1 antigen, a mutated p53 antigen, a mutated Ras antigen, a ERBB2 (HER2) antigen, a folate binding protein (FBP) antigen, a B7-H6 antigen, a CD123 antigen, a LI CAM (CD 171) antigen, a CD30 antigen, a CD33 antigen, a CD37 antigen, a CD38 antigen, a CD4 antigen, a CD47 antigen, a CD70 antigen, a CD73 antigen, a CD79b antigen, a CLL1 (CD371) antigen, a CEA antigen, a Claudin 18.1 antigen, a Claudin 18.2 antigen, a CS-1 antigen, a CSPG4 antigen, aEFGRvIII antigen, aENPP3 antigen, aEpCAM antigen, a EphA2 antigen, a ErbB antigen, a FAP antigen, a FRa antigen, a GD3 antigen, a Glypican 3 antigen, a IL-llRa antigen, a LewisY/LeY antigen, a MUC-16 antigen, a NGFR antigen, a PD1 antigen, a PD-Ll antigen, a PSCA antigen, a PSMA antigen, a RORl antigen, a SLAMF7 antigen, a TACI antigen, a TAG72 antigen, a ULBP antigen, a MICA/B antigen, a VEGFR2 antigen, a WT1 antigen, a PTK7 antigen, a STEAP1 antigen, a STEAP2 antigen, a PD-L2 antigen, a c-Met antigen, a CD70 antigen, a MG7 antigen, a EGFR1 antigen, a PDGFR1/2 antigen, aNectin-4 antigen, a CD133 antigen, aDNAM-1 antigen, aFLT-3 antigen, a ULBP 1-6 antigen, a CD 112 antigen, a CD44 antigen, or a TROP2 antigen. The transmembrane domain can be a T cell receptor a chain transmembrane domain, a T cell receptor b chain transmembrane domain, a CD3z chain transmembrane domain, a CD28 transmembrane domain, a CD3e transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD8 transmembrane domain, a CD9 transmembrane domain, a CD 16 transmembrane domain, a CD22 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD 134 transmembrane domain, a CD 137 transmembrane domain, a CD278 transmembrane domain, a CD 154 transmembrane domain, a CD357 transmembrane domain, a CD2 transmembrane domain, a CD6 transmembrane domain, a CD1 la transmembrane domain, a CDllc transmembrane domain, a CD 14 transmembrane domain, a CD 18 transmembrane domain, a CD20 transmembrane domain, a CD25 transmembrane domain, a CD30 transmembrane domain, a CD32 transmembrane domain, a CD34 transmembrane domain, a CD40 transmembrane domain, a CD43 transmembrane domain, a CD47 transmembrane domain, a CD49 transmembrane domain, a CD52 transmembrane domain, a CD56 transmembrane domain, a CD57 transmembrane domain, a CD58 transmembrane domain, a CD69 transmembrane domain, a CD70 transmembrane domain, a CD94 transmembrane domain, a CD95 transmembrane domain, a CD 123 transmembrane domain, a CD 133 transmembrane domain, a CD138 transmembrane domain, a CD 152 transmembrane domain, a CD 159a transmembrane domain, a CD223 transmembrane domain, a CD274 transmembrane domain, a CD276 transmembrane domain, a CD279 transmembrane domain, a CD314 transmembrane domain, a CD366 transmembrane domain, or a CD371 transmembrane domain. The transmembrane domain can be a CD8 transmembrane domain. The STAP domain can be a SLP76 STAP domain, a Gads STAP domain, a LAT STAP domain, a TRAF1 STAP domain, a TRAF2 STAP domain, a TRAF3 STAP domain, a TRAF5 STAP domain, a Grb2 STAP domain, a SOS STAP domain, a NEMO STAP domain, or a TAB 1/2 STAP domain. The phosphatase domain can be a Src homology region 2 domain- containing phosphatase- 1 (SHP-1) phosphatase domain or a Src homology region 2 domain- containing phosphatase-2 (SHP-2) phosphatase domain.

In another aspect, this document features T cells comprising one or more CARs an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain.

In another aspect, this document features nucleic acid constructs comprising a nucleic acid sequence encoding a CAR including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain. The nucleic acid sequence encoding the CAR can include a CD8a secretion signal (e.g., a CD8a secretion signal). The nucleic acid construct also can include a promoter operably linked to the nucleic acid sequence encoding the CAR. The promoter can be a U6 promoter, a HI promoter, a RSV LTR promoter, a CMV promoter, a SV40 promoter, a dihydrofolate reductase promoter, a b-actin promoter, a PGK promoter, an MND promoter, an EFla promoter, a MND promoter, or a mCAG promoter. The nucleic acid sequence encoding the CAR can include a polyadenylation signal.

In another aspect, this document features T cells comprising a nucleic acid construct encoding a CAR including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain, where the nucleic acid construct expresses the CAR.

In another aspect, this document features pharmaceutical compositions comprising T cells comprising one or more CARs an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain and/or T cells comprising a nucleic acid construct encoding a CAR including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain, where the nucleic acid construct expresses the CAR; and a pharmaceutically acceptable excipient.

In another aspect, this document features methods for treating a mammal having cancer. The methods can include or consist essentially of administering to a mammal having cancer T cells comprising one or more CARs an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain and/or T cells comprising a nucleic acid construct encoding a CAR including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain, where the nucleic acid construct expresses the CAR; where the CAR targets an antigen expressed by the cancer. The mammal can be a human. The cancer can be prostate cancer, ovarian cancer, cervical cancer, colorectal cancer, intestinal cancer, testicular cancer, skin cancer, lung cancer, thyroid cancer, bone cancer, breast cancer, bladder cancer, uterine cancer, vaginal cancer, pancreatic cancer, liver cancer, kidney cancer, brain cancer, spinal cord cancer, oral cancer, parotid tumor, ALL, AML, CLL, CML, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, myeloma, soft tissue sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, Ewing sarcoma, osteosarcoma, GIST, penile cancer, or cervical cancer. The administering can include intravenous injection.

In another aspect, this document features methods for treating a mammal having fibrosis. The methods can include or consist essentially of administering to a mammal having fibrosis T cells comprising one or more CARs an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain and/or T cells comprising a nucleic acid construct encoding a CAR including an extracellular component comprising an antigen binding domain, a transmembrane domain; and an intracellular component comprising a phosphatase domain and a STAP domain, where the nucleic acid construct expresses the CAR; where the CAR targets an antigen expressed by a fibrotic cell within the mammal. The mammal can be a human. The administering can include intravenous injection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a schematic of an exemplary SPRTS-CAR provided herein. Figures 2 A and 2B: Schematic diagrams of nucleic acid molecules encoding signal producing T cell stimulator CARs (SPRTS-CAR). Figure 2A depicts a schematic of nucleic acid molecule encoding a canonical SPRTS-CAR. Figure 2B depicts a schematic of a nucleic acid molecule encoding an exemplary SPRTS-CAR targeting ROR1.

Figures 3 A and 3B depict a schematic of an exemplary SPRTS CAR comprising one or more intracellular Src Homology 2 (SH2) domains and a kinase domain.

Figure 4 depicts a schematic of cells comprising a SPRTS-CAR and a CAR.

Figure 5 depicts a the expression of SPRTS-CARs introduced into human primary T cells.

DETAILED DESCRIPTION

This document provides CARs (e.g, SPRTS-CARs) that include: (a) an intracellular component containing a signal transduction domain (e.g, a kinase domain or a phosphatase domain) and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain as well as CAR-T cells presenting one or more CARs provided herein. In some cases, CAR-T cells presenting one or more CARs provided herein can be used to stimulate a signal transduction pathway (e.g., a signal transduction pathway that involves the signal transduction domain of the CAR) in cells (e.g, cells within a mammal such as a human) thereby inducing one or more cellular responses in the cells. In some cases, CAR-T cells presenting one or more CARs provided herein can be used for treating a mammal (e.g, a human) having, or at risk of developing, cancer.

A signal transduction pathway domain of an intracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include any appropriate signal transduction pathway domain or combination of signal transduction pathway domains (e.g, a combination of two, three, or four signal transduction pathway domains). In some cases, the signal transduction pathway domain of an intracellular component of a CAR provided herein is not a T cell signaling domain. For example, the signal transduction pathway domain of an intracellular component of a CAR provided herein is not an intracellular signaling domain normally found within T cells or NK cells (e.g., a CD3-zeta (Q domain).

In some cases, a signal transduction pathway domain of an intracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include a kinase domain. As used herein, a kinase domain is a polypeptide (e.g, a kinase polypeptide), or a biologically active fragment thereof, that can phosphorylate a target polypeptide to stimulate a signal transduction pathway thereby inducing one or more cellular responses (e.g, induction of cell growth, cell proliferation, metabolism, secretion of cytokines and/or chemokines, cytotoxicity toward tumor cells to which the CAR binds, cell differentiation, expression of regulatory receptors on the cell surface, and cell senescence).

In some cases, a kinase domain can be (or can be derived from) a polypeptide that can be present in (e.g, is typically present in) a lipid raft. In some cases, a kinase domain can be (or can be derived from) a polypeptide that can be activated by clustering of polypeptides involved in the signal transduction pathways. In some cases, a kinase domain can be (or can be derived from) a polypeptide in the Src-family of kinases. In some cases, a kinase domain can be (or can be derived from) a polypeptide in the Tec-family of kinases. In some cases, a kinase domain can be (or can be derived from) a polypeptide in the ZAP-70-family of kinases. In some cases, a kinase domain can be (or can be derived from) a tyrosine kinase (e.g, a receptor tyrosine kinase). Examples of kinase domains that can be used in an intracellular component of a CAR provided herein include, without limitation, kinase domains from lymphocyte specific protein tyrosine kinase (Lck) polypeptides, kinase domains from Z chain-associated protein kinase 70 (ZAP-70) polypeptides, kinase domains from interleukin-2-inducible T cell tyrosine kinase (ITK) polypeptides, kinase domains from receptor-interacting protein kinase (RIP) polypeptides (e.g, RIP kinase (RIPK) 1 polypeptides, RIPK2 polypeptides, RIPK3 polypeptides, RIPK4 polypeptides, and RIPK5 polypeptides), kinase domains from IKB kinase (IKK) polypeptides (e.g, IKK-a (IKKl) polypeptides, IKK-b (IKK2) polypeptides, and IKK-g (NEMO) polypeptides), kinase domains from apoptosis signal-regulating kinase 1 (ASK-1) polypeptides (also referred to as mitogen-activated protein kinase 5 (MAP3K5) polypeptides), kinase domains from extracellular signal-regulated kinase (ERK) polypeptides (e.g, ERK1 polypeptides, ERK2 polypeptides, ERK3/4 polypeptides, ERK5 polypeptides, and ERK7 polypeptides), kinase domains from NF-kappa-B-inducing kinase (NIK) polypeptides (also referred to as mitogen- activated protein kinase 14 (MAP3K14)), kinase domains from MEKK1 polypeptides (also referred to as MAP3K1), kinase domains from Src polypeptides, kinase domains from Yes polypeptides, kinase domains from Lyn polypeptides, kinase domains from Fyn polypeptides, kinase domains from Fgr polypeptides, kinase domains from Yrk polypeptides, kinase domains from Btk polypeptides, kinase domains from Bmx polypeptides, kinase domains from Txk polypeptides, kinase domains from Tec polypeptides, kinase domains from Syk polypeptides, kinase domains from FAK polypeptides, kinase domains from Pyk2 polypeptides, kinase domains from Csk polypeptides, kinase domains from EGFR polypeptides, kinase domains from PDGFR polypeptides, and biologically active fragments thereof.

In some cases, a signal transduction pathway domain of an intracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include a phosphatase domain. As used herein, a phosphatase domain is a polypeptide (e.g, a phosphatase polypeptide), or a biologically active fragment thereof, that can dephosphorylate a target polypeptide to stimulate a signal transduction pathway thereby inducing one or more cellular responses (e.g, repression of cell growth, cell proliferation, metabolism, secretion of cytokines and/or chemokines, cytotoxicity toward tumor cells to which the CAR binds, cell differentiation, expression of regulatory receptors on the cell surface, and cell senescence). In some cases, a phosphatase domain can be (or can be derived from) a polypeptide that can be present in (e.g, is typically present in) a lipid raft. In some cases, a phosphatase domain can be (or can be derived from) a polypeptide that can be activated by clustering of polypeptides involved in the signal transduction pathways. Examples of phosphatase domains that can be used in an intracellular component of a CAR provided herein include, without limitation, phosphatase domains from Src homology region 2 domain-containing phosphatase- 1 (SHP-1) polypeptides, phosphatase domains from Src homology region 2 domain-containing phosphatase-2 (SHP-2) polypeptides, and biologically active fragments thereof.

In some cases, the signal transduction pathway domain of an intracellular component of a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be as described elsewhere (see, e.g., Rohrs etal. , iScience , 23:101023 (2020); Aaronson, Science, 254(5035): 1146-53 (1991); Aaronson et al., Adv. Exp. Med. Biol., 348:7- 22 (1993); Rosen et al, Oncologist, 15(3):216-35 (2010); Yablonski etal., Front. Immunol., 10:1704 (2019); and Clark etal, eLife, 8:e45789 (2019)).

A STAP domain of an intracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include any appropriate STAP domain or combination of STAP domains (e.g, a combination of two, three, or four signal transduction pathway domains). In some cases, the STAP domain of an intracellular component of a CAR provided herein is not a T cell co-stimulatory domain. For example, the STAP domain of an intracellular component of a CAR provided herein is not a co stimulatory domain normally found within T cells or NK cells (e.g, a CD28 domain, a CD27 domain, a CD134 (0X40) domain, and a CD137 (4-1BB) domain).

As used herein, a STAP domain is a polypeptide (e.g, a STAP polypeptide), or a biologically active fragment thereof, that can bind a signal transduction pathway polypeptide thereby mediating protein-protein interactions to facilitate the formation of larger signaling complexes. In some cases, a STAP domain can have reduced enzymatic activity (e.g, as compared to a polypeptide from which the STAP domain is derived). In some cases, a STAP domain can lack any enzymatic activity (e.g, as compared to a polypeptide from which the STAP domain is derived). Examples of STAP domains that can be used in an intracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) include, without limitation, STAP domains from SH2-domain-containing leukocyte protein of 76 kDa (SLP76) polypeptides, STAP domains from Grb2-family adaptor downstream of She (Gads) polypeptides, STAP domains from linker of activation of T cells (LAT) polypeptides, STAP domains from TNF receptor associated factor (TRAF) polypeptides ( e.g ., TRAF1 polypeptides, TRAF2 polypeptides, TRAF3 polypeptides, and TRAF5 polypeptides), STAP domains from growth factor receptor-bound protein 2 (Grb2) polypeptides, STAP domains from son of sevenless (SOS) polypeptides, STAP domains from NF-kappa-B essential modulator (NEMO) polypeptides, STAP domains from TGF-beta- activated kinase (TAB) polypeptides, STAP domains from TAB2 polypeptides, and biologically active fragments thereof.

A transmembrane domain of a CAR provided herein (e.g., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include any appropriate transmembrane domain. A transmembrane domain can be located between an intracellular component of a CAR provided herein and an extracellular component of a CAR provided herein. In some cases, a transmembrane domain can provide structural stability for the CAR. For example, a transmembrane domain can include a structure (e.g, a hydrophobic alpha helix structure) that can span a cell membrane and can anchor the CAR to the cell membrane. In some cases, a transmembrane domain can be derived from (e.g, can include, consist essentially of, or consist of at least a part of) the transmembrane region(s) of a membrane-bound polypeptide (e.g, a transmembrane polypeptide). In some cases, a transmembrane domain can be a synthetic transmembrane domain. In some cases, a transmembrane domain can be a type I receptor transmembrane domain. In some cases, a transmembrane domain can be a type II receptor transmembrane domain. Examples of transmembrane domains that can be used as described herein include, without limitation, T cell receptor a chain (TCRa) transmembrane domains, T cell receptor b chain (TCRP) transmembrane domains, CD3 transmembrane domains (e.g, CD3-zeta (Q transmembrane domains and CD3 epsilon (e) transmembrane domains), CD4 transmembrane domains, CD8 transmembrane domains (e.g, a CD8a transmembrane domains and CD8P transmembrane domains), CD28 transmembrane domains, CD 16 transmembrane domains, CD45 transmembrane domains, CD5 transmembrane domains, CD9 transmembrane domains, CD22 transmembrane domains, CD33 transmembrane domains, CD37 transmembrane domains, CD64 transmembrane domains, CD80 transmembrane domains, CD86 transmembrane domains, CD 134 transmembrane domains, CD 137 transmembrane domains, CD278 transmembrane domains, CD 154 transmembrane domains, CD357 transmembrane domains, CD2 transmembrane domains, CD6 transmembrane domains, CDlla transmembrane domains, CDllc transmembrane domains, CD 14 transmembrane domains, CD 18 transmembrane domains, CD20 transmembrane domains, CD25 transmembrane domains, CD30 transmembrane domains, CD32 transmembrane domains, CD34 transmembrane domains, CD40 transmembrane domains, CD43 transmembrane domains, CD47 transmembrane domains, CD49 transmembrane domains, CD52 transmembrane domains, CD56 transmembrane domains, CD57 transmembrane domains, CD58 transmembrane domains, CD69 transmembrane domains, CD70 transmembrane domains, CD94 transmembrane domains, CD95 transmembrane domains, CD 123 transmembrane domains, CD 133 transmembrane domains, CD138 transmembrane domains, CD 152 transmembrane domains, CD 159a transmembrane domains, CD223 transmembrane domains, CD274 transmembrane domains, CD276 transmembrane domains, CD279 transmembrane domains, CD314 transmembrane domains, CD366 transmembrane domains, and CD371 transmembrane domains.

In some cases, a transmembrane domain of a CAR provided herein ( e.g ., a SPRTS- CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2016/0120906 such as U.S. Patent Application Publication No. 2016/0120906 at paragraphs [0155], [0161], [0269], Figure 4, and Figure 11; U.S. Patent Application Publication No. 2019/0209616 such as U.S. Patent Application Publication No. 2019/0209616 at paragraph [0026]; U.S. Patent Application Publication No. 2018/0000914 such as U.S. Patent Application Publication No. 2018/0000914 at paragraphs [0168] - [0171]; U.S. Patent Application Publication No. 2017/0183418 such as U.S. Patent Application Publication No. 2017/0183418 at paragraphs [0116] - [0118]; U.S. Patent Application Publication No. 2017/0183413 such as U.S. Patent Application Publication No. 2017/0183413 at paragraphs [0116] - [0118]; and U.S. Patent Application Publication No. 2017/0145094 such as U.S. Patent Application Publication No. 2017/0145094 at paragraphs [0104] - [0107]

An extracellular component of a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include any appropriate antigen binding domain. In some cases, an antigen-binding domain can include an antibody or a fragment thereof that targets an antigen (e.g., a cancer antigen such as a CD 19 polypeptide). Examples of antigen-binding domains include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), and single domain camelid antibody binding domains (VHH). In some cases, an antigen-binding domain can target (e.g, can target and bind to) a tumor-specific antigen. For example, a CAR provided herein can express (e.g, can be engineered to express) an antigen binding domain that can bind to a tumor-specific antigen (e.g, an antigen present on cancer cells with minimal, or no, expression on non-cancerous cell types).

An extracellular component of a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include an antigen binding domain that can recognize any appropriate antigen. In some cases, an antigen-binding domain in an extracellular component of a CAR provided herein can target (e.g, can target and bind to) a tumor-specific antigen (e.g, an antigen present on cancer cells with minimal, or no, expression on non-cancerous cell types). For example, an antigen-binding domain in an extracellular component of a CAR provided herein can bind to a tumor-specific antigen (e.g, a cell surface tumor-specific antigen) expressed by a cancer cell in a mammal having, or at risk of developing, cancer. Examples of antigens that can be recognized by a CAR provided herein include, without limitation, antigens from cluster of differentiation 19 (CD 19) polypeptides, antigens from CD22 polypeptides, antigens from CD20 polypeptides, antigens from GD2 polypeptides, antigens from EGFRvIII polypeptides, antigens from mesothelin polypeptides, antigens from IL-13RA polypeptides, antigens from B-cell maturation antigen (BCMA) polypeptides, antigens from CD138 polypeptides, antigens from NKG2-D polypeptides, antigens from HER2/Neu polypeptides, antigens from IL-13RA2 polypeptides, antigens from CD 137 polypeptides, antigens from CD28 polypeptides, antigens from B7-H3 (CD276) polypeptides, antigens from CD16V polypeptides, antigens from CA-125 polypeptides, antigens from MUC-1 polypeptides, antigens from epithelial tumor antigen polypeptides, antigens from melanoma-associated antigen polypeptides, antigens from mutated p53 polypeptides, antigens from mutated Ras polypeptides, antigens from ERBB2 (HER2) polypeptides, antigens from folate binding protein (FBP) polypeptides, antigens from B7-H6 polypeptides, antigens from CD123 polypeptides, antigens from L1CAM (CD 171) polypeptides, antigens from CD30 polypeptides, antigens from CD33 polypeptides, antigens from CD37 polypeptides, antigens from CD38 polypeptides, antigens from CD4 polypeptides, antigens from CD47 polypeptides, antigens from CD70 polypeptides, antigens from CD73 polypeptides, antigens from CD79b polypeptides, antigens from CLL1 (CD371) polypeptides, antigens from CEA polypeptides, antigens from Claudin 18.1 polypeptides, antigens from Claudin 18.2 polypeptides, antigens from CS-1 polypeptides, antigens from CSPG4 polypeptides, antigens from EFGRvIII polypeptides, antigens from ENPP3 polypeptides, antigens from EpCAM polypeptides, antigens from EphA2 polypeptides, antigens from ErbB polypeptides, antigens from FAP polypeptides, antigens from FRa polypeptides, antigens from GD3 polypeptides, antigens from Glypican 3 polypeptides, antigens from IL-llRa polypeptides, antigens from LewisY/LeY polypeptides, antigens from MUC-16 polypeptides, antigens from NGFR polypeptides, antigens from PD1 polypeptides, antigens from PD-L1 polypeptides, antigens from PSCA polypeptides, antigens from PSMA polypeptides, antigens from ROR1 polypeptides, antigens from SLAMF7 polypeptides, antigens from TACI polypeptides, antigens from TAG72 polypeptides, antigens from ULBP polypeptides, antigens from MICA/B polypeptides, antigens from VEGFR2 polypeptides, antigens from WT1 polypeptides, antigens from PTK7 polypeptides, antigens from STEAPl polypeptides, antigens from STEAP2 polypeptides, antigens from PD-L2 polypeptides, antigens from c-Met polypeptides, antigens from CD70 polypeptides, antigens from MG7 polypeptides, antigens from EGFR1 polypeptides, antigens from PDGFR1/2 polypeptides, antigens from Nectin-4 polypeptides, antigens from CD 133 polypeptides, antigens from DNAM-1 polypeptides, antigens from FLT-3 polypeptides, antigens from ULBP1-6 polypeptides, antigens from CD112 polypeptides, antigens from CD44 polypeptides, and antigens from TROP2 polypeptides.

In some cases, an antigen-binding domain of a CAR provided herein ( e.g ., a SPRTS- CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2017/0183418 such as U.S. Patent Application Publication No. 2017/0183418 at paragraph [0015] and the sequence listing; U.S. Patent Application Publication No. 2017/0183413 such as U.S. Patent Application Publication No. 2017/0183413 at paragraph [0049], Figure 2, Table 9, and the sequence listing; U.S. Patent Application Publication No. 2018/0291079 such as U.S. Patent Application Publication No. 2018/0291079 at paragraphs [0041] - [0045], and Table 4; U.S. Patent Application Publication No. 2020/0289563 such as U.S. Patent Application Publication No.

2020/0289563 at paragraphs [0006] - [0053], [0186] - [0189], and Table 1; and U.S. Patent Application Publication No. 2003/0211097 such as U.S. Patent Application Publication No. 2003/0211097 at paragraphs [0081] and [0211-0215] and the sequence listing.

In some cases, a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include a hinge region. A hinge region, when present, can be in any appropriate location within a CAR provided herein. In some cases, a hinge region present in a CAR provided herein can be between transmembrane domain of a CAR provided herein and an extracellular component of a CAR provided herein. In some cases, a hinge region can provide a CAR with increased flexibility for the antigen-binding domain. For example, a hinge region can reduce spatial limitations of an antigen-binding domain of a CAR and its target antigen ( e.g ., to increase binding between an antigen-binding domain of a CAR and its target antigen). In some cases, a hinge region can be a synthetic hinge region. For example, a synthetic hinge region can include a repeating sequence of amino acids. For example, a synthetic hinge region can include a sequence of identical amino acids. Examples of hinge regions that can be used as described herein include, without limitation, a membrane-proximal region from an IgG, a membrane-proximal region from CD8, and a membrane-proximal region from CD28. In some cases, a hinge region of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018/0000914 such as U.S. Patent Application Publication No. 2018/0000914 at paragraph [0168], and Table 1; U.S. Patent Application Publication No. 2017/0183418 such as U.S. Patent Application Publication No. 2017/0183418 at paragraphs [0034], [0037], [0040], and Table 2; U.S. Patent Application Publication No. 2017/0183413 such as U.S. Patent Application Publication No. 2017/0183413 at paragraph [0116]; and U.S. Patent Application Publication No. 2017/0145094 such as U.S. Patent Application Publication No. 2017/0145094 at paragraph [0104]).

In some cases, a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include a secretion signal. A secretion signal, when present, can be in any appropriate location within a CAR provided herein. In some cases, a secretion signal present in a CAR provided herein can be in an extracellular component of a CAR provided herein. Examples of secretion signals that can be used as described herein include, without limitation, a secretion signal from CD8 (e.g, CD8a) and a secretion signal from CD28. In some cases, a secretion signal of a CAR can be as described elsewhere (see, e.g, U.S. Patent Application Publication No. 2018/0000914 such as U.S. Patent Application Publication No. 2018/0000914 at paragraph [0408]; U.S. Patent Application Publication No. 2017/0183418 such as U.S. Patent Application Publication No. 2017/0183418 at paragraphs [0234] and [0238]; U.S. Patent Application Publication No. 2017/0183413 such as U.S. Patent Application Publication No. 2017/0183413 at paragraph [0157]; and U.S. Patent Application Publication No. 2017/0145094 such as U.S. Patent Application Publication No. 2017/0145094 at paragraph [0209]).

In some cases, a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include one or more linkers. A linker, when present, can be in any appropriate location within a CAR provided herein. In some cases, a linker present in a CAR provided herein can be between transmembrane domain of a CAR provided herein and an intracellular component of a CAR provided herein. In some cases, a linker present in a CAR provided herein can be between transmembrane domain of a CAR provided herein and an extracellular component of a CAR provided herein. In some cases, a linker can be rich in small and/or polar amino acids such as Gly and Ser. In some cases, a linker can provide flexibility to a CAR provided herein. In some cases, a linker can provide solubility to a CAR provided herein. A linker that can be included in a CAR provided herein can be any length. Examples of linkers that can be included in a CAR provided herein include, without limitation, a linker including the amino acid sequence GSG, a linker including the amino acid sequence GGS, a linker including the amino acid sequence G3S (SEQ ID NO: 1), a linker including the amino acid sequence G4S (SEQ ID NO:2), a linker including the amino acid sequence G5S (SEQ ID NO:3), a linker including the amino acid sequence (GGS) ₇ (SEQ ID NO:4), a linker including the amino acid sequence AAA, a linker including the amino acid sequence (G ₃ S) ₃ (SEQ ID NO:5), a linker including the amino acid sequence (G ₄ S) ₃ (SEQ ID NO:6), a linker including the amino acid sequence(G ₄ S) ₄ (SEQ ID NO:7), a linker including the amino acid sequence (G ₄ S)s (SEQ ID NO:8), and a linker including the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 9).

In some cases, a CAR provided herein (e.g., a SPRTS-CAR) comprises: (a) an intracellular component containing a kinase domain or a phosphatase domain and one or more SH2 domain; (b) a transmembrane domain; and (c) an extracellular component containing a hinge domain, and one or more scFvs. In some cases, the kinase domain is adjacent the one of more SH2 domains, in some cases the kinase domain is flanked by the one of more SH2 domains. In some cases, the SH2 and kinase domains are derived from the same protein (e.g., ZAP70 or LCK). In some cases, the SH2 and kinase domains are derived from different proteins (e.g., ZAP70, LCK, SLAP, etc.). In some cases, the SH2 and kinase domains are derived from a combination of the same and different proteins. Exemplary sequences for these embodiments are provided as SEQ ID NOs.: 24-29 (nucleic acids) and SEQ ID NOs.: 30-35 (polypeptides).

In some cases where a CAR-T cell expresses two CARs, the first CAR is a SPRTS- CAR comprising one or more STAP signaling domains, and the second CAR comprises a CD3 signaling domain. In some cases, the SPRTS-CAR and the CAR target the same antigen. In some cases, the SPRTS-CAR and the CAR target different antigens. In cases where a CAR-T cell express a SPRTS-CAR and a CAR targeting an antigen presented by a cancer cell (e.g., a tumor-specific antigen), binding of the SPRTS-CAR and the CAR to the antigen drives an increased synergistic immune response compared to a CAR-T cells expressing only a SPRTS-CAR or only a CAR.

This document also provides CAR-T cells expressing one or more (e.g, one, two, three, four, or more) CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain). One or more CARs provided herein can be expressed in (e.g, and presented on the surface of) any type of T cell. In some cases, a T cell can be a naive T cell. In some cases, a T cell can be a stimulated T cell. Examples of T cells that can be designed to express a CAR provided herein include, without limitation, naive T cells (e.g, CD4 ⁺ naive T cells and/or CD8 ⁺ naive T cells), cytotoxic T cells (e.g, CD4 ⁺ CTLs and/or CD8 ⁺ CTLs), tissue resident memory T cells, and central memory T cells. In some cases, a population of T cells can be obtained from a mammal (e.g, a mammal having, or at risk of developing, cancer). For example, a population of T cells (e.g, a natural T cell population) can be obtained from a mammal to be treated using the materials and methods described herein. In some cases, a population of T cells can be obtained from a donor mammal (e.g, a donor mammal of the same species) as the mammal to be treated using the materials and methods described herein. For example, when treating a human, a population of T cells can be obtained from a donor human. In some cases, when treating a human, a population of T cells can be obtained from a donor transgenic pig donor that was engineered to be compatible with humans. In cases where a donor mammal and the mammal to be treated using the materials and methods described herein are humans, the donor human and the human to be treated using the materials and methods described herein can present the same or similar human leukocyte antigens (HLAs; e.g, can be HLA-matched).

In some cases, one or more (e.g, one, two, three, four, or more) CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be expressed in an immune cell other than a T cell. For example, this document also provides CAR-NK cells expressing one or more (e.g, one, two, three, four, or more) CARs provided herein.

In cases where a CAR-T cell expresses two or more (e.g, two, three, four, or more) CARs provided herein (e.g, two or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain), the CARs can be the same or can be different.

In some cases where a CAR-T cell expresses two or more different CARs, the CARs (e.g, a first CAR and a second CAR) can activate the same signal transduction pathway or can activate different signal transduction pathways. For example, a CAR-T cell expressing two or more different CARs that can activate the same signal transduction pathway, the different CARs can be stimulated during CAR clustering (e.g, CAR clustering within the CAR-T cell).

In some cases where a CAR-T cell expresses two or more different CARs, the CARs (e.g, a first CAR and a second CAR) can target different antigens. For example, a CAR-T cell expressing two or more different CARs can express a first CAR that can target an antigen presented by a cancer cell (e.g, a tumor-specific antigen) and can activate a T cell response, and can express a second CAR that can target an antigen presented by a healthy (e.g, non-cancerous) cell and can suppress a T cell response and/or can suppress the signal transduction pathway activated by the first CAR. In cases where a CAR-T cell expressing a first CAR that can target an antigen presented by a cancer cell ( e.g ., a tumor-specific antigen) and can activate a T cell response, and expressing a second CAR that can target an antigen presented by a healthy (e.g., non-cancerous) cell and can suppress a T cells response and/or can suppress the signal transduction pathway activated by the first CAR is engaged only by an antigen presented by a cancer cell binding to the first CAR, the T cell can be activated to target (e.g, target and destroy) the cancer cell. In cases where a CAR-T cell expressing a first CAR that can target an antigen presented by a cancer cell (e.g, a tumor-specific antigen) and can activate a T cell response, and expressing a second CAR that can target an antigen presented by a healthy (e.g, non-cancerous) cell and can suppress a T cells response and/or can suppress the signal transduction pathway activated by the first CAR is engaged by an antigen presented by a cancer cell binding to the first CAR and is engaged by an antigen presented by a healthy cell binding to the second CAR, the T-cell is not activated thereby reducing or eliminating the T-cell from targeting healthy (e.g, non-cancerous) cells.

Any appropriate method can be used to express one or more CARS provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) on the surface of a T cell as described herein. For example, a nucleic acid encoding a CAR provided herein can be introduced into one or more T cells. A nucleic acid encoding a CAR provided herein can be introduced in a T cell using any appropriate method. In some cases, a nucleic acid encoding a CAR provided herein can be introduced into a T cell by transduction (e.g, viral transduction using a retroviral vector such as a lentiviral vector) or transfection. In some cases, a nucleic acid encoding a CAR provided herein can be introduced ex vivo into one or more T cells. For example, ex vivo engineering of T cells expressing a CAR provided herein can include transducing isolated T cells with a lentiviral vector encoding a CAR provided herein. In cases where T cells are engineered ex vivo to express a CAR provided herein, the T cells can be obtained from any appropriate source (e.g, a mammal such as the mammal to be treated or a donor mammal). In some cases, a nucleic acid encoding a CAR provided herein that is introduced into a T cell can be transient (e.g, does not integrate into the T cell genome). In some cases, a nucleic acid encoding a CAR provided herein that is introduced into a T cell can be stable ( e.g ., is integrated into the T cell genome).

This document also provides constructs (e.g., nucleic acid constructs) encoding a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain). In some cases, a construct encoding a CAR provided herein can be an expression vector (e.g, a cDNA expression vector). In some cases, a construct encoding a CAR provided herein can be a viral vector (e.g, vectors based on a virus such as adenoviruses, adeno-associated viruses, retroviruses, and lentiviruses).

In addition to nucleic acid encoding a CAR provided herein (e.g, a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain), a construct (e.g. , a non-viral vector or a viral vector) can contain one or more regulatory elements operably linked to the nucleic acid encoding a CAR provided herein. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g, transcription or translation) of a nucleic acid. The choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a construct to facilitate transcription of a nucleic acid encoding a CAR provided herein. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be constitutive or inducible (e.g, in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a CAR provided herein in a general or cell/tissue- specific manner. Examples of promoters that can be used to drive expression of a CAR provided herein in cells include, without limitation, U6 promoters, HI promoters, RSV promoters (optionally, with the RSV enhancer), CMV promoters (optionally, with the CMV enhancer), SV40 promoters, dihydrofolate reductase promoters, b-actin promoters, PGK promoters, EF la promoters, MND promoters, and mCAG promoters. As used herein, “operably linked” refers to positioning of a regulatory element in a vector relative to a nucleic acid encoding a CAR provided herein in such a way as to permit or facilitate expression of the encoded CAR. For example, a construct can contain a promoter and nucleic acid encoding a CAR provided herein. In this case, the promoter is operably linked to a nucleic acid encoding a CAR provided herein such that it drives expression of the CAR polypeptide in cells.

In some cases, a nucleic acid construct encoding a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can encode (e.g., can be designed to encode) a CAR including: (a) an intracellular component containing a kinase domain from a ZAP-70 polypeptide and a STAP domain from a SLP76 polypeptide; (b) a transmembrane domain from a CD28 polypeptide; and (c) an extracellular component containing an antigen binding domain that can recognize an antigen from a ROR1 polypeptide (a ROR1 antigen).

This document also provides methods for using CARs provided herein (e.g, SPRTS- CARs including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain). In some cases, CAR-T cells presenting one or more CARs provided herein can be used to modulate one or more cellular responses (e.g, cell growth, cell proliferation, metabolism, secretion of cytokines and/or chemokines, cytotoxicity toward tumor cells to which the CAR binds, cell differentiation, expression of regulatory receptors on the cell surface, and cell senescence) within a cell. For example, CAR-T cells presenting one or more CARs provided herein can be administered (e.g, by adoptive cell transfer) to a mammal to modulate one or more cellular responses of cells within that mammal.

In some cases, CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be used to treat a mammal ( e.g ., a human) having, or at risk of developing, cancer. For example, CAR-T cells presenting one or more CARs provided herein can be administered (e.g., by adoptive cell transfer) to a mammal in need thereof (e.g, a mammal such as a human having, or at risk of developing, cancer) to treat the mammal.

Any appropriate mammal having, or at risk of developing, cancer can be treated as described herein. For example, humans, non-human primates (e.g, monkeys), horses, bovine species, porcine species, dogs, cats, mice, rats, and camelids can be treated as described herein. In some cases, a human having, or at risk of developing, cancer can be administered CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain).

In some cases, the methods described herein also can include identifying a mammal as having, as being at risk of developing, cancer. Examples of methods for identifying a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g, blood, urine, bone marrow aspirates, and/or cerebral spinal fluid aspirates), biopsy, imaging tests (e.g, X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g, bone scans), endoscopy, and/or genetic tests.

A mammal (e.g, a human) having, at risk of developing, cancer, can be administered or instructed to self-administer CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain).

CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be administered to a mammal by any appropriate route. For example, CAR-T cells presenting one or more CARs provided herein can be administered locally or systemically. In some cases, CAR-T cells presenting one or more CARs provided herein can be designed for parenteral (e.g, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intrathecal, intratumoral, hepatic arterial, and intraparenchymal catheterization) administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The materials and methods described herein can be used to treat a mammal ( e.g ., a human) having any type of cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer.

In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can express a tumor-specific antigen (e.g., an antigenic substance produced by a cancer cell). Examples of cancers that can be treated as described herein include, without limitation, prostate cancers, ovarian cancers, cervical cancers, colorectal cancers, intestinal cancers, testicular cancers, skin cancers, lung cancers, thyroid cancers, bone cancers, breast cancers, bladder cancers, uterine cancers, vaginal cancers, pancreatic cancers, liver cancers, kidney cancers, brain cancers, spinal cord cancers, oral cancers, parotid tumors, leukemias (e.g, acute lymphocytic leukemias (ALLs), acute myeloid leukemias (AMLs), chronic lymphocytic leukemias (CLLs), and chronic myeloid leukemias (CMLs)), lymphomas (e.g, Hodgkin’s lymphomas and non-Hodgkin’s lymphomas), myelomas, soft tissue sarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas,

Ewing sarcomas, osteosarcomas, gastrointestinal stromal tumors (GISTs), penile cancers, and cervical cancers.

In some cases, the materials and methods provided herein can be used to reduce or eliminate the number of cancer cells present within a mammal (e.g, a human) having, or at risk of developing, cancer. For example, a mammal in need thereof (e.g, a mammal having, or at risk of developing, cancer) can be administered CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) to reduce or eliminate the number of cancer cells present within the mammal. For example, the materials and methods described herein can be used to reduce the number of cancer cells present within a mammal having, or at risk of developing, cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to reduce the size ( e.g ., volume) of one or more tumors present within a mammal having, or at risk of developing, cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the number of cancer cells present within a mammal being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a mammal is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a mammal.

In some cases, the materials and methods provided herein can be used to improve survival of a mammal (e.g., a human) having, or at risk of developing, cancer. For example, a mammal in need thereof (e.g, a mammal having, or at risk of developing, cancer) can be administered CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) to improve survival of the mammal. For example, the materials and methods described herein can be used to improve the survival of a mammal having, or at risk of developing, cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to improve the survival of a mammal having, or at risk of developing, cancer by, for example, at least 6 months (e.g, about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

In some cases, CAR-T cells presenting one or more CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be used as the sole active agent to treat a mammal ( e.g ., a human) having, or at risk of developing, cancer.

In some cases, CAR-T cells presenting one or more CARs provided herein (e.g., one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can be administered to a mammal (e.g, a human) having, or at risk of developing, cancer together with one or more (e.g, one, two, three, four, five or more) additional agents/therapies used to treat cancer. Examples of anti-cancer agents include, without limitation, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab (e.g, AVASTIN ^® ), olaparib, rucaparib, niraparib, cetuximab, trastuzumab (e.g, HERCEPTIN ^® ), nivolumab (e.g., OPDIVO ^® ), Rituxan, pembrolizumab (e.g, KEYTRUDA ^® ), panitumumab, and any combinations thereof. In cases where CAR-T cells presenting one or more CARs provided herein are used in combination with additional agents used to treat cancer, the one or more additional agents can be administered at the same time (e.g, in a single composition containing both CAR-T cells presenting one or more CARs provided herein and the one or more additional agents) or independently. For example, CAR-T cells presenting one or more CARs provided herein can be administered first, and the one or more additional agents administered second, or vice versa. Examples of therapies that can be used to treat cancer include, without limitation, surgery, and radiation therapy. In cases where CAR-T cells presenting one or more CARs provided herein are used in combination with one or more additional therapies used to treat cancer, the one or more additional therapies can be performed at the same time or independently of the administration of CAR-T cells presenting one or more CARs provided herein. For example, the CAR-T cells presenting one or more CARs provided herein can be administered before, during, or after the one or more additional therapies are performed.

In some cases, the materials and methods described herein can be used to treat a mammal having a disease, disorder, or condition other than cancer. For example, a mammal having a disease, disorder, or condition other than cancer can be administered CAR-T cells presenting one or more CARs provided herein (e.g., one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain). In such cases, the T cells can be designed to express one or more CARs provided herein that include an antigen binding domain that can target an antigen associated with a disease, disorder, or condition. For example, T cells can be designed to express one or more CARs provided herein that include an antigen binding domain that can target an antigen associated with an autoimmune disease. For example, T cells can be designed to express one or more CARs provided herein that include an antigen binding domain that can target an antigen associated with an inflammatory diseases. For example, T cells can be designed to express one or more CARs provided herein that include an antigen binding domain that can target an antigen associated with a monogenic disease. Examples of diseases, disorders or conditions other than cancer that can be treated as described herein include, without limitation, fibrosis, multiple sclerosis, systemic lupus erythematosus, pemphigus vulgaris, lupus nephritis, myasthenia gravis, Sjogren syndrome, vasculitis, idiopathic thrombocytopenic purpura, neuromyelitis optica spectrum disorders, anti-N-methyl D-aspartate (NMD A) receptor (anti-NMDAR) encephalitis, rheumatoid arthritis, osteoarthritis, celiac disease, polymyalgia rheumatica, ankylosing spondylitis, Type 1 diabetes, temporal arteritis, and alopecia areata. For example, a mammal having fibrosis can be administered CAR-T cells presenting one or more CARs (e.g, one or more SPRTS-CARs) each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain that can target an antigen associated with fibrosis.

This document also provides kits containing one or more (e.g, one, two, three, or more) CARs provided herein (e.g, one or more SPRTS-CARs each including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain). In some cases, a kit can include T cells presenting one or more CARs provided herein. For example, T cell presenting one or more CARs provided herein can be combined with packaging material to form a kit. In some cases, a kit can include one or more constructs ( e.g ., nucleic acid constructs) encoding a CAR provided herein. For example, one or more constructs (e.g., nucleic acid constructs) encoding a CAR provided herein can be combined with packaging material to form a kit. The packaging material included in such a kit typically contains instructions or a label describing how the composition can be used. For example, a kit can contain instructions and/or a label describing how T cells presenting one or more CARs provided herein can be used in an adoptive cell transfer to treat a mammal having, or at risk of developing, cancer as described herein. For example, a kit can contain instructions and/or a label describing how a construct (e.g, a nucleic acid construct) encoding a CAR provided herein can be used to express the CAR in a T cell to engineer CAR-T cells presenting one or more CARs provided herein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Design, cloning, viral vector production, and T Cell transduction of a SPRTS- CAR insertion cassette

This Example illustrates in silico design and cloning of a SPRTS-CAR insertion cassette and introduction into the TRAC locus of T cells. Not all of the following steps are required nor must the order of the steps be as presented.

The nucleotide sequence encoding the SPRTS-CAR is codon-optimized for expression in mammalian cells using the IDT codon optimizer tool (Integrated DNA Technologies, Inc., Coralville, IA). The oligonucleotide sequence coding for the SPRTS- CAR fusion protein can be provided to a commercial manufacturer for synthesis.

Example 2: Preparation of cytotoxic T cells (CD 4+ and CD8+) from peripheral blood mononuclear cells (PBMCs)

This Example illustrates the preparation of CD4+ and CD8+ T cells from donor peripheral blood mononuclear cells (PBMCs). CD4+ and CD8+ T cells were prepared from donor PBMCs essentially as follows. T cells were isolated from peripheral blood mononuclear cells (PBMCs) using RoboSep-S (STEMCELL Technologies) and EasySep™ Human T cell Isolation Kit (STEMCELL Technologies 1795 lRF) and activated for 3 days in the presence of anti-CD3/CD28 beads (Dynabeads™; Gibco 11132D) in ImmunoCult-XF complete medium (ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies 10981), CTS Immune Cell SR (Gibco A2596102), Antibiotics-Antimycotics (100X, Corning 30-004-Cl)) supplemented with recombinant human (rh) IL-2 (100 units/mL). After 3 days, beads were removed via magnetic separation and cells were expanded for 1 day in ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL).

Example 3: Cloning, Expression, Production, and Assembly of Casl2a Guide/nucleoprotein Complexes

This Example describes a method for cloning, expressing, and purifying Casl2a guide/nucleoprotein complexes, as well as methods of producing Casl2a guide components.

A. Cloning of a Casl2 protein

The Acidaminococcus spp. (strain BV3L6) catalytically active Casl2a protein sequence was codon optimized for expression in E. coli cells. At the C-terminus, one nuclear localization sequence (NLS) was added. Oligonucleotide sequences coding for the Casl2a- NLS protein (referred to as the AsCasl2a and Casl2a protein in the following Examples) were provided to commercial manufacturers for synthesis. DNA sequences were then cloned into suitable bacterial expression vectors using standard cloning methods.

B. Expression and purification of a Casl2a protein

The AsCasl2a protein was expressed in E. coli using an expression vector and purified using affinity chromatography, ion exchange, and size exclusion chromatography, essentially as described in, for example, Swarts et al. (Molecular Cell, 2017, 66:221-233). C. Production of Casl2a guide components

Casl2a guides were produced by linking a targeting region to a particular Casl2a guide activating region. A targeting region, or spacer, preferably comprised a 20-nucleotide target binding sequence. The target binding sequence was complementary to a target sequence that occurred downstream (in a 3’ direction) of a 5’- TTTV or 5’ - TTTN PAM.

Cas 12a guide sequences (such as crRNAs and chRDNA) were provided to a commercial manufacturer for synthesis.

Guide RNA components (such as crRNAs) can be produced by in vitro transcription ( e.g ., T7 Quick High Yield RNA Synthesis Kit; New England Biolabs, Ipswich, MA) from double-stranded (ds) DNA templates by incorporating a T7 promoter at the 5’ end of the dsDNA template sequences.

D. Assembly of a Casl2a guide/nucleoprotein complex

Acidaminococcus spp. Casl2a (AsCasl2a) was tagged at the C-terminus with a nuclear localization sequence (NLS) and was recombinantly expressed in E. coli and purified using chromatographic methods. Nucleoprotein complexes were formed at a concentration of 80 pmol Casl2a protein:240 pmol guide, unless otherwise stated. Prior to assembly with Casl2a protein, each of the guide components (e.g., crRNA or chRDNA) was adjusted to the desired total concentration (240 pmol) in a final volume of 1 mΐ, incubated for 2 minutes at 95°C, removed from a thermocycler, and allowed to equilibrate to room temperature. The Casl2a protein was diluted to an appropriate concentration in binding buffer (60mM TRIS- acetate, 150 mM potassium acetate, 30 mM magnesium acetate, at pH 7.9) to a final volume of 1.5 mΐ and mixed with the 1 mΐ of the guide components, followed by incubation at 37°C for 10 minutes.

Example 4: Nucleofection of T Cells (CD4+ and CD8+) from PBMCs with Casl2a Guide/Nucleoprotein Complexes

This Example describes the nucleofection of activated T cells with a Casl2a guide/nucleoprotein complex.

The Cas 12a guide/nucleoprotein complexes of Example 2 were transfected into primary activated T cells (CD4+ and CD8+) (prepared as described in Example 1) using the NucleofectorTM 96-well Shuttle System (Lonza, Allendale, NJ). The Casl2a guide/nucleoprotein complex were dispensed in a 2.5 mΐ final volume into individual wells of a 96-well plate. The suspended T cells were pelleted by centrifugation for 10 minutes at 200 x g, washed with calcium and magnesium-free phosphate buffered saline (PBS), and the cell pellet was resuspended in 10 ml of calcium and magnesium-free PBS. The cells were counted using the Countess® II Automated Cell Counter (Life Technologies; Grand Island, NY).

Approximately 2.2e7 cells were transferred to a 15 ml conical tube and pelleted. The PBS was aspirated, and the cells resuspended in NucleofectorTM P4 (Lonza, Allendale, NJ) solution to a density of 2e5-le6 cells/ml per sample. Twenty (20) mΐ of the cell suspension was then added to each well containing 2.5 mΐ of the Casl2a guide/nucleoprotein complexes, and the entire volume from each well was transferred to a well of a 96-well NucleocuvetteTM Plate (Lonza, Allendale, NJ). The plate was loaded onto the NucleofectorTM 96-well Shuttle (Lonza, Allendale, NJ) and cells nucleofected using the CA137 NucleofectorTM program (Lonza, Allendale, NJ). Post-nucleofection, 77.5 mΐ of ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL) was added to each well, and the entire volume of transfected cell suspension was transferred to a 96-well cell culture plate containing 100 mΐ pre-warmed ImmunoCult-XF complete medium supplemented with IL-2 (100 units/mL). The plate was transferred to a tissue culture incubator and maintained at 37°C in 5% CO2 for 48 hours before downstream analysis.

Example 5: Cloning of AA V Donor Cassette, AA V Production, and AA V Transduction of Primary Cells

This Example describes the design and cloning of a DNA donor cassette into an AAV vector, production of AAV, delivery of Casl2a chRDNA guide/nucleoprotein complexes into primary cells, and transduction of primary cells with AAV for site-specific integration of a SPRTS-CAR expression cassette into primary cells.

AAV can be engineered to deliver DNA donor polynucleotides to mammalian cells.

If AAV delivery is combined with a genomic cleavage event, and the DNA donor polynucleotide in the AAV is flanked by homology arms, the DNA donor polynucleotide can be seamlessly inserted into the genomic cut site by HDR, as described in, for example, Eyquem etal. (Nature, 2017, 543:113-117).

A. In silico design of AAV donor cassettes and rAAV production

The SPRTS-CAR construct is designed to contain an N-terminal secretion signal (CD8a signal peptide), a scFv portion specific for BCMA, and a c-terminal BGH polyadenylation signal sequence. A mammalian promoter sequence is inserted upstream of the SPRTS-CAR polynucleotide. In order to site-specifically insert DNA donor polynucleotides into the host cell genome after site-specific cleavage, a target site is chosen in the endogenous TRAC locus. Then, 500 bp long homology arms 5’ and 3’ of the cut site are identified. The 5’ and 3’ homology arms are appended to the end of the DNA donor polynucleotides, wherein the DNA donor polynucleotides are orientated in a reverse orientation (i.e., 3’ to 5’) relative to the homology arms.

Oligonucleotide sequences coding for DNA donor polynucleotides are provided to a commercial manufacturer for synthesis into a suitable recombinant AAV (rAAV) plasmid. A rAAV plasmid is provided to a commercial manufacturer for packaging into AAV6 viruses.

B. Primary T cell transduction with rAAV

Primary activated T cells are obtained from PBMCs as described in Example 2. Casl2a chRDNA guide/nucleoprotein complexes targeting the genes encoding TRAC are prepared as described in Example 2. T cells are transfected with TRAC -targeting Casl2a chRDNA guide/nucleoprotein complexes, and between 1 minute and 4 hours after nucleofection, cells are infected with the AAV6 virus packaged with SPRTS-CAR donor sequence at an MOI of 1 x 10 ⁶ . T cells are cultured in ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/mL) for 24 hours after the transductions. The next day, the transduced T cells are transferred to 50 mL conical tubes and centrifuged at 300 x g for approximately 7-10 minutes to pellet cells. The supernatant is discarded, and the pellet is gently resuspended, and the T cells pooled in an appropriate volume of ImmunoCult-XF complete medium (STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/mL). The enumerated T cells are resuspended at 1 x 106 cells/mL in ImmunoCult-XF complete medium STEMCELLS Technologies, Cambridge, MA) supplemented with IL-2 (100 units/mL), and plated into as many T-175 suspension flasks as required (max volume per flask is 250 mL).

C. Expression of anti-BCMA SPRTS-CAR cells

In vitro characterization of anti-BCMA, SPRTS-CAR cells, as well as control (TRAC knock out (KO)) and wild type T cells, is performed 7 days after transduction.

CAR-T cells are evaluated for expression via FACS for either anti-BCMA SPRTS- CAR expression using recombinant BCMA protein conjugated to phycoerythrin (PE); expression of TRAC using an anti-TCR a/b specific antibody conjugate to Alexa Fluor® 647 (ThermoFisher Scientific, Waltham, MA).

D. In vitro cytotoxicity of anti-BCMA SPRTS-CAR cells

The cytotoxicity of anti-BCMA SPRTS-CAR cells is evaluated in vitro against a multiple myeloma NCI-H929 cells line, which present the BCMA antigen. TRAC KO T cells are used as a control for SPRTS-CAR -mediated killing. Briefly, target cells (NCI-H929 (T)) are labeled with CellTrace™ Violet (CTV; Thermo Fisher C34557) to distinguish them from effector anti-BCM, SPRTS-CAR , and cells are co-cultured at E:T ratios of 0: 1, 1 :20, 1:10, 1:5, 1:3, 1:1, 3:1, and 10:1 (3 co-culture wells/E:T ratio). Cytotoxicity is measured by gating on CTV cell population (target cells) and live cells as measured by propidium iodide (PI) after 48 hours in co-culture. Data is analyzed by flow cytometry (Intellicyt iQue Screener Plus). Specific lysis is calculated using the following equation for each well: Specific lysis = 1- (number of live target cells in test sample/number of live target cells in control sample).

The methods presented herein can be used to manufacture other cells using Casl2a chRDNA guide molecules for the site-specific introduction of donor polynucleotides comprising a chimeric antigen receptor (CAR). Additional donor polynucleotides expressing non-CAR polypeptides (i.e., SPRTS-CAR fusions construct) can be similarly introduced using the guidance herein. Example 6: Kinase Domains for use in SPRTS-CAR-T Cells to Modulate Cellular Responses

An exemplary LCK kinase domain that can be used in a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 10.

SEQ ID NO: 10

MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPP ASPLQ DNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSL EP EPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEW KHYKIRNL DNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLK LV ERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAW TQE PIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANI LV SDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTE IV THGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVL ED FFTATEGQYQPQP

An exemplary ZAP-70 kinase domain that can be used in a CAR provided herein (e.g., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 11.

SEQ ID NO: 11

MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFH HFPIE RQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAM VR DYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTD GK FLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGL I Y CLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPM PM DTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKV LK QGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREE IP VSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYT AR SAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGK RM ECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAAC A

An exemplary ITK kinase domain that can be used in a CAR provided herein (e.g., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 12.

SEQ ID NO: 12

MNNFILLEEQLIKKSQQKRRTSPSNFKVRFFVLTKASLAYFEDRHGKKRTLKGS IELSRIKC VEIVKSDISIPCHYKYPFQVVHDNYLLYVFAPDRESRQRWVLALKEETRNNNSLVPKYHP NF WMDGKWRCCSQLEKLATGCAQYDPTKNASKKPLPPTPEDNRRPLWEPEETW IALYDYQTND PQELALRRNEEYCLLDSSEIHWWRVQDRNGHEGYVPSSYLVEKSPNNLETYEWYNKS ISRDK AEKLLLDTGKEGAEMVRDSRTAGTYTVSVFTKAW SENNPCIKHYHIKETNDNPKRYYVAEK YVFDSIPLLINYHQHNGGGLVTRLRYPVCFGRQKAPVTAGLRYGKWVIDPSELTFVQEIG SG QFGLVHLGYWLNKDKVAIKTIREGAMSEEDFIEEAEVMMKLSHPKLVQLYGVCLEQAPIC LV FEEMEHGCLSDYLRTQRGLFAAETLLGMCLDVCEGMAYLEEACVIHRDLAARNCLVGENQ VI KVSDFGMTRFVLDDQYTSSTGTKFPVKWASPEVFSFSRYSSKSDVWSFGVLMWEVFSEGK IP YENRSNSEW EDISTGFRLYKPRLASTHVYQIMNHCWKERPEDRPAFSRLLRQLAEIAESGL

An exemplary RIP kinase domain that can be used in a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 13. SEQ ID NO: 13

MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHN EALLE EAKMMNRLRHSRVVKLLGVI IEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIE GMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLAS FKMWSKLNNEEHNELREVDGTAKKN GGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAI FANKEPYENAICEQQLIMCIKSGNRPD VDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEY SN ENAW KRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEP SLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNT EG KGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIP SH MPSLHNIPVPETNYLGNTPTMPFSSLPPTDES IKYTIYNSTGIQIGAYNYMEIGGTSSSLLD STNTNFKEEPAAKYQAIFDNTTSLTDKHLDPIRENLGKHWKNCARKLGFTQSQIDEIDHD YE RDGLKEKVYQMLQKWVMREGIKGATVGKLAQALHQCSRIDLLSSLI YVSQN

Example 7: Regulatory Elements to Express a Nucleic Acid Encoding a CAR SPRTS-CAR

A nucleic acid encoding a CAR provided herein ( e.g. , a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include an MND promoter that can comprise, consist essentially of, or consist of the nucleic acid sequence of SEQ ID NO: 14.

MND promoter (SEQ ID NO: 14) tttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagct ag gatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaag ca gttcctgccccggctcagggccaagaacagttggaacagcagaatatgggccaaacagga ta tctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcgg tc ccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaa at gaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgctt ct gctccccgagctcaataaaagagcccacaacccctcactcggcgcgacgcgtcataGCCA CC A nucleic acid encoding a CAR provided herein ( e.g ., a SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain) can include a polyadenylation (poly A) signal that can comprise, consist essentially of, or consist of the nucleic acid sequence of SEQ ID NO: 15.

BGH PolyA (SEQ ID NO: 15)

CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA CCCTG

GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAG

TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG GGAAG

ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

Example 8: SPRTS-CAR Amino Acid Sequence

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a CD8A (1-21) signal peptide that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16.

SEQ ID NO: 16

MALPVTALLLPLALLLHAARP

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a BCMA PMC306 scFv that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 17

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYI IPYNDATKYNE KFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSSGGGG SG GGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYYA SQ SITGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPPTFGGGTKVEIK

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a CD8A hinge (22-182) that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO : 18.

SEQ ID NO: 18

SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKP KAAEG LDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS IMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a CD8 transmembrane domain (183-203) that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 19.

SEQ ID NO: 19

IYIWAPLAGTCGVLLLSLVIT

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a STAP domain of the SLP76 polypeptide (422-530) that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:20.

SEQ ID NO:20

WYVSYITRPEAEAALRKINQDGTFLVRDSSKKTTTNPYVLMVLYKDKVYNIQIRYQK ESQVY LLGTGLRGKEDFLSVSDIIDYFRKMPLLLIDGKNRGSRYQCTLTHAA

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a Zap- 70 SH2 domain (163-254) that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:21.

SEQ ID NO:21

WYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAG KYCIP EGTKFDTLWQLVEYLKLKADGLIYCLKEAC

An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can include a Zap- 70 kinase domain (338-600) that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:22.

SEQ ID NO:22

LIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLD NPYIV RLIGVCQAEALMLVMEMAGGGPLHKFLVGKREE IPVSNVAELLHQVSMGMKYLEEKNFVHRD LAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSD VW SYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWI YKWEDRPD FLTVEQRMRACYYSL An exemplary SPRTS-CAR including: (a) an intracellular component containing a kinase domain or a phosphatase domain and a STAP domain; (b) a transmembrane domain; and (c) an extracellular component containing an antigen binding domain can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO:23.

SEQ ID NO:23

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKK PGSSVKVSCKASGYTFTSYVMHWVRQAP GQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNY DG YFDVWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQS ISDY LHWYQQKPGQAPRLLIYYASQSITGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGH SF PPTFGGGTKVEIKAAASQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAA SP TFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS IMYFSHFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAG TCGVLLLSLVITWYVSYITRPEAEAALRKINQDGTFLVRDSSKKTTTNPYVLMVLYKDKV YN IQIRYQKESQVYLLGTGLRGKEDFLSVSDI IDYFRKMPLLLIDGKNRGSRYQCTLTHAAWYH SSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLI YGKTVYHYLISQDKAGKYCIPEGT KFDTLWQLVEYLKLKADGLIYCLKEACLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKV LK QGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREE IP VSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYT AR SAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGK RM ECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL

CD8A signal peptide (amino acids 1-21 of the CD8 protein)

BCMA PMC306 scFv AAA hinge linker

CD8A hinge (amino acids 22-182 of the CD8 protein)

CD8 Transmembrane domain (amino acids 183-203 of the CD8 protein) SLP76 STAP domain (amino acids 422-530 of the SLP76 protein) Zap-70 SH2 (amino acids 163-254 of the Zap-70 protein)

Zap-70 Protein Kinase (amino acids 338-600 of the Zap-70 protein) Example 9: Using SPRTS-CAR-T Cells to Modulate Cellular Responses

SPRTS-CAR-T Cells including: (a) an intracellular component containing a kinase domain from a ZAP-70 polypeptide and a STAP domain from a SLP76 polypeptide; (b) a transmembrane domain from a CD28 polypeptide; and (c) an extracellular component containing an antigen binding domain that can recognize an antigen from a ROR1 polypeptide (a ROR1 antigen) are contacted with cells overexpressing a ROR1 polypeptide. In some cases, one or more ROR1 antigens that can be recognized the SPRTS-CAR-T Cells also can be provided to the SPRTS-CAR-T Cells that are in contact with the cells overexpressing a ROR1 polypeptide. Upon binding of a ROR1 antigen, the SPRTS-CAR-T Cells are activated to stimulate the ZAP-70 signal transduction pathway to induce T cells to differentiate, proliferate, and secrete a number of cytokines. In some cases, an increase in ZAP-70 signal transduction can be confirmed and/or monitored by detecting phosphorylation of a LAT polypeptide, a SLP76 polypeptide, a p38 MAP kinase polypeptide, and/or a Lck polypeptide.

Example 10: Using SPRTS-CAR-T Cells to Treat Cancer

A human identified as having cancer that includes cancer cells overexpressing a ROR1 polypeptide is administered SPRTS-CAR-T Cells including: (a) an intracellular component containing a kinase domain from a ZAP-70 polypeptide and a STAP domain from a SLP76 polypeptide; (b) a transmembrane domain from a CD28 polypeptide; and (c) an extracellular component containing an antigen binding domain that can recognize an antigen from a ROR1 polypeptide (a ROR1 antigen). Upon binding of a ROR1 antigen, the administered SPRTS-CAR-T Cells are activated to target ( e.g. , target and destroy) cancer cells presenting the ROR1 antigen.

Example 11: Introducing a SPRTS-CAR construct into human T cells and expression of the SPRTS-CAR in the T cell This example describes the transfection and transduction of activated human T cells with a Casl2a guide/nucleoprotein complex and an AAV6 virus comprising a SPRTS-CAR with an intracellular region comprising a dual SH2 domain and a kinase domain.

A. Design of AAV donor cassettes and rAAV production

The SPRTS-CAR constructs are designed essentially as described in Example 5A, with the modification that the intracellular C-terminus of the SPRTS-CAR is engineered to comprise two SH2 domains and a kinase domain.

Designs of the six SPRTS-CARs are provided in Tables 1 and 2.

Table 1. Sequence of the SPRTS-CAR constructs

Table 2. Structure of the SPRTS-CARs

*AA: amino acid; SS: signal sequence, Prom: promoter, TM: transmembrane domain, Term: terminator

The six SPRTS-CARs shown in Tables 1 and 2 were provided to a commercial manufacturer for synthesis and packaging into AAV6 capsid. B. Transfection and transduction of human T cells with AAV6 containing SPRTS- CARs

The Casl2a/guide nucleoprotein complexes (NPC) of Example 2 targeting the TRAC locus were transfected into primary activated T cells (CD4+ and CD8+) (prepared as described in Example 1) using the method described in Example 4.

AAV6 constructs containing SPRTS-CARs corresponding to SEQ ID NO. 25, SEQ ID NO. 26, and SEQ ID NO. 29 were transduced into T cells within 1 hour of NPC transfection. TRAC knock-out (KO)-only and wild-type T cell controls were included.

Four days after the transfection-transduction, cells were stained for expression of the anti-BCMA scFv using the method of Example 5. SPRTS-CAR expression is shown in FIG. 5 and Table 3 below. The data represents the average of three biological replicates (dashed line in FIG. 5 represents the limit of detection based on TRAC KO and wild-type controls).

Table 3. SPRTS-CAR expression in primary T-cells

The results presented in FIG. 5 and Table 3 demonstrate the transduction of human T cells and subsequent expression of the SPRTS-CAR construct described in this application.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.