CHIMERIC ANTIGEN RECEPTOR THAT BINDS MESOTHELIN

Field of the invention

This invention was made with government support under Grant Number CA193140 and CA016086 awarded by the National Institutes of Health and award number W81XWH-18-1- 0441 from the U.S. Department of Defense. The government has certain rights in the invention.

The present invention relates to chimeric antigen receptors (CARs) comprising a human variable heavy chain (VH) single domain antibody that binds to mesothelin (MSLN) and which has the CDR1 ,2 and 3 sequences as defined herein. Such CAR can be used in the treatment of cancer, e.g. for the treatment of solid tumors. CARs are able to redirect immune cell specificity and reactivity toward a selected target exploiting the ligand-binding domain properties. The present invention also relates to a CAR with an antigen binding domain comprising a VH single domain antibody that binds to mesothelin and which has sequences as defined herein and also a VH single domain antibody that binds prostate-specific membrane antigen (PSMA).

The present invention further relates to polynucleotides encoding a CAR of the invention, vectors encoding such polypeptides and isolated cells expressing the CAR of the invention at their surface. Such cells can be used in therapy, in particular for the treatment of cancer e.g. for the treatment of solid tumors. Methods for treating disease, for example cancer, are thus also within the scope of the invention.

Introduction

Adoptive cellular therapy (ACT) has received much attention as a technique for cancer treatment. One therapeutic approach of ACT involves genetic engineering of T cells to express chimeric antigen receptors (CARs) on the surface of T cells to enable targeting of specific tumours. Once the CAR is expressed in T cells, the CAR modified T cell (CAR-T or CAR- T cell) acquires properties that include antigen-specific recognition, activation and proliferation and the cells thus act as “living drugs”. The purpose of expressing a CAR in a T cell is therefore to redirect immune reactivity of the cell to a chosen target. Furthermore, CARs with different strength and signalling can also modulate T cell expansion as well as alter the strength of T cell activation.

CARs are synthetic receptors typically consisting of a targeting/binding moiety that is associated with one or more signaling domains in a single fusion molecule. The binding moiety of a CAR typically consists of an antigen-binding domain of a single-chain antibody (scFv) comprising paired antibody light chain and heavy chain variable domains (VL and VH) that are fused into a single polypeptide chain via a short flexible linker ^11-13 . The scFv retains the same specificity and a similar affinity as the full antibody from which it was derived and is capable of binding to the specific target of interest. In addition to an extracellular antigen-binding domain CARs also comprise a transmembrane domain and signaling molecules such as costimulatory endodomains and CD3 chain ^1-3 .

CARs combine antigen-specificity and T cell activating properties in a single fusion molecule.

First generation CARs typically included the cytoplasmic region of the CD3zeta or Fc receptor y chain as their signalling domain. First generation CARs have been tested in phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, where they have induced modest responses (reviewed references 4 and 5 and in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009). Second generation CARs, which contain the signalling domains of both CD28 and CD3zeta, provide dual signalling to direct combined activating and co-stimulatory signals. Third generation CARs are more complex with three or more signalling domains ^{4 5} .

CARs including single domain antibodies that binds PSMA have been described in WO2017/191476 (incorporated herein by reference).

Expression of CARs in T cells enables specific targeting of surface antigens in a Major Histocompatibility Complex-independent manner and associated T cell activation ⁴ ’ ⁵ . While classical CARs use scFvs as the antigen-binding moiety, other ligands fused with signaling molecules of the T-cell receptor (TCR) complex can also trigger phosphorylation events in T cells ⁶ . For example, engineering of natural receptors such as NKG2D and CD27 fused with CD3 zeta have been shown to redirect T cell specificity ^{7 8} . Ligands to receptors such as IL- 13Ra2 have also been engineered to redirect T cell specificity towards glioblastoma ⁹ . More recently, synthetic antigen binding moieties as exemplified by a ‘monobody’ based on the type III domain of fibronectin (FN3) have also been shown to serve as a robust platform to generate CAR molecules ¹⁰ . Therefore, utilizing alternative binding moieties to replace scFvs to generate CAR remains a critical area because scFvs are frequently unstable and showing intrinsic tendency to self-aggregation, which may lead to tonic signaling and loss of function of CAR-T cells in vivo' CAR-Ts that have scFv can also form clusters on the membrane due to crosslinking of heavy and light chains of different CAR-Ts. This results in unwanted constitutive signalling. Furthermore, scFv used in CAR-Ts may be derived from mouse mAbs and thus have potential immunogenicity issues as the anti-scFv response not only initiates T cell response by potentially crosslinking but also eradicates CAR-Ts from circulation. Thus, scFv have a number of characteristics that can have a negative impact on the therapeutic efficacy of CAR-Ts. In particular, non-specific activation of T cells is a major safety concern.

Without wishing to be bound by theory, these problems are likely to be compounded when scFvs are used to design CAR-Ts having bispecific, bivalent or biparatopic antigen binding moieties.

Heavy-chain-only antibodies (HcAbs) without light chains have been reported in camelids and cartilaginous fish ^14-16 and have shown to exhibit strong and specific antigen binding ¹² . Single domains targeting BCMA have been developed to generate BCMA-specific CAR-T cells for the treatment of multiple myeloma ¹⁸ . Whilst scFv typically used in CARs have the potential for unwanted aggregation, cluster formation and immunogenicity, the use of human VH domains provides a stable format with substantially reduced potential for immunogenicity, aggregation or unfolding. This is particularly useful when designing CAR-Ts having bispecific, bivalent or biparatopic antigen binding moieties. As demonstrated by the inventors herein, multiple human VH domains can readily be used in such mutimeric format thus facilitating the generation of multispecific CARs that enable simultaneous targeting of more than one target or epitope.

Whether human derived VH only domains can be used as a CAR to target antigens expressed in solid tumors is unknown. Treatment failure and/or disease recurrence after CAR-T cell therapy can be caused by epitope or antigen loss ¹⁰ . In particular, the inherently heterogeneous expression pattern of antigens in solid tumors can easily cause tumor escape after targeted immunotherapy ¹⁰ ’ ¹⁹²⁰ . Therefore, targeting multiple tumor-associated antigens (TAAs) is generally expected to improve the outcome of CAR-T cell therapy in solid tumor ^{10 19} . However, including multiple scFvs within a CAR causes protein instability and decreases binding specificity and affinity. The VH domain-only format of CARs provides an ideal solution for multiple antigen targeting because VH domains are smaller in size and may more easily fold in the correct 3D structure compared to scFv molecules.

Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. It is however highly expressed in several human cancers including malignant mesothelioma, pancreatic, ovarian, endometrial and lung adenocarcinoma. In the context of cancer, high expression levels of MSLN have been correlated with poor prognosis in ovarian cancer, cholangiocarcinoma, lung adenocarcinoma and triple-negative breast cancer. The limited expression of mesothelin on normal human tissues and high expression in several human cancers makes mesothelin an attractive candidate for cancer therapy. Several therapeutic strategies have been designed for targeting mesothelin on tumor cells including tumor vaccine strategy, antibody-based therapies and adoptive CAR T-cell therapy. These therapies are being evaluated in phase I and/or phase II clinical trials.

Therefore, the present invention is aimed at mitigating the shortcomings of existing CAR-T therapies using mesothelin antibodies by providing CARs and CAR-Ts with a single VH domain antibody targeting mesothelin as defined herein.

Summary of the Invention

The invention relates to CARs that target human mesothelin (MSLN) and comprises an antigen binding domain that includes a single VH domain antibody with CDR1 ,2 and 3 sequences as described herein. The inventors have shown that such VH domain-based CARs exhibited comparable or superior antitumor activity compared to traditional CARs incorporating scFv. Moreover, the inventors have demonstrated that such VH domains are particularly suitable for constructing bispecific CAR-T cells that can significantly better control tumors with heterogeneous antigen expression.

In one aspect, the invention provides an isolated chimeric antigen receptor (CAR) comprising a VH single domain antibody that specifically binds to human mesothelin (MSLN), wherein the VH single domain antibody comprises a CDR1 comprising SEQ NO. 4 or a sequence with 1 , 2 or 3 amino acid modifications, a CDR2 comprising SEQ NO. 5 or a sequence with 1 , 2, 3 or 4 amino acid modifications and a CDR3 comprising SEQ NO. 6 or a sequence with 1 , 2 or 3 amino acid modifications. In one embodiment, the antigen binding domain of the CAR further comprises a moiety that binds PSMA, for example a VH single domain antibody.

In another aspect, the invention provides an isolated nucleic acid encoding a CAR as described above.

In another aspect, the invention provides a vector comprising a nucleic acid as described above.

In another aspect, the invention provides a host cell comprising a nucleic acid or a vector as described above.

In another aspect, the invention provides an isolated cell or cell population comprising one or more CAR according, a nucleic acid or a vector as described above. In another aspect, the invention provides a pharmaceutical composition comprising a cell or cell population as described above and a pharmaceutical acceptable carrier, excipient or diluent.

In another aspect, the invention provides a method for treating a cancer comprising administering a cell or cell population or a pharmaceutical composition as described above.

In another aspect, the invention provides a cell or cell population or a pharmaceutical composition as above for use in therapy, e.g. for use in the treatment of cancer.

In another aspect, the invention provides a use of a cell or cell population or a pharmaceutical composition as above in the manufacture of a medicament for the treatment of cancer. In another aspect, the invention provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of a cell or cell population as above or a pharmaceutical composition as above.

In another aspect, the invention provides a method of providing an anti-tumor immunity in a subject, the method comprising administering to the subject an effective amount of a cell or cell population as above or a pharmaceutical composition as above.

In another aspect, the invention provides an ex vivo or in vitro method for generating a population of cells for use in adaptive immunotherapy comprising transforming said cell with a nucleic acid encoding a CAR as above, a nucleic acid as above or a vector as above. In another aspect, the invention provides a method for imaging tumour binding comprising expressing a CAR as above in a cell wherein said CAR comprises a label.

In another aspect, the invention provides a kit comprising a CAR, a nucleic acid or a vector or a cell or cell population as above.

In another aspect, the invention provides a combination therapy comprising an effective amount of a cell or cell population or a pharmaceutical composition as above and an effective amount of an immune checkpoint inhibitor.

In another aspect, the invention provides a use of a VH single domain antibody comprising a CDR1 comprising SEQ NO. 1 or a sequence with 1 , 2 or 3 amino acid substitutions, CDR2 comprising SEQ NO. 2 or a sequence with 1 , 2, 3 or 4 amino acid substitutions and a CDR3 comprising SEQ NO. 3 or a sequence with 1 , 2 or 3 amino acid in a CAR. In another aspect, the invention provides a method of making a population of cells as above, the method comprising:

(i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells;

(ii) contacting the population of cells (for example, T cells) with the nucleic acid molecule of encoding a CAR as described above, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and

(iii) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration.

Figures

The invention is further described in the following non-limiting figures.

Figure 1. Human antibody VH domain-based CAR targeting PSMA is expressed and signals in T cells. (A) Schematic diagram of J591 and PSMA-VH constructs. (B, C) Representative flow cytometry plots (B) and summary (C) illustrating J591 and PSMA-VH expression in T cells. The CD19-specific CAR (CD19) and non-transduced T cells (NT) were used as positive and negative controls, respectively. ****p < 0.0001 , One-way ANOVA. (D) In vitro expansion of CD19, J591 , PSMA-VH and NT T cells; error bars represent SD, (n = 4). p >0.05 by One-way ANOVA. (E) T cell subset composition based on CD45RA and CCR7 expression in CD19, J591 , PSMA-VH and NT T cells at day 14 of culture; error bars represent SD, (n = 4). p >0.05 by One-way ANOVA. (F) Western blots detecting phosphorylation of CAR- CD3 , Akt and ERK in J591 and PSMA-VH T cells activated via CAR cross-linking with an anti- FLAG Ab followed by incubation with a secondary Ab to induce the aggregation of CAR molecules. Total CAR. CD3 and endogenous CD3 were used as loading controls. Data are representative of 2 experiments.

Figure 2. T cells expressing the human antibody VH domain-based CAR targeting PSMA are functional in vitro. (A) Representative flow cytometry plots showing the expression of PSMA in C4-2, PC3 and PC3 cells engineered with a retroviral vector to express PSMA. (B, C) Representative flow cytometry plots (B) and summary (C) illustrating Granzyme-B expression of T cells expressing either J591 or PSMA-VH cocultured overnight with a tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2; error bars represent SD, (n = 4). p >0.05 by t-test. (D, E) Representative flow cytometry plots (D) and summary (E) illustrating the kinetics of CD69 expression of T cells expressing either J591 or PSMA-VH and cocultured overnight with a tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2. Data are representative of 4 experiments. ***p < 0.001 Two-way ANOVA. (F) Representative flow cytometry plots showing coculture of CD19, J591 and PSMA-VH T cells with C4-2-eGFP, PC3-eGFP and PC3-PSMA-eGFP. T cells were cocultured with tumor cells at an E:T ratio of 1 :5 for 6 days. At day 6, all cells were collected and analyzed by flow cytometry to quantify tumor cells (GFP) and T cells (CD3), respectively. (G) Summary of coculture of CD19, J591 and PSMA-VH T cells with tumor cells in (F); error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (H, I) IFN-y (H) and IL-2 (I) were detected by ELISA in the coculture supernatant of cocultures of CD19, J591 and PSMA-VH T cells with tumor cells illustrated in (F); error bars represent SD, (n = 5). *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , Two-way ANOVA. (J) Representative flow cytometry plots showing the proliferation of J591 and PSMA-VH T cells in response to tumor cells as assed by CFSE dilution. Data are representative of 4 experiments.

Figure 3. T cells expressing the human antibody VH domain-based CAR targeting PSMA are functional in vivo. (A) Schematic of the metastatic prostate cancer model using PC3- PSMA-FFIuc-eGFP tumor cells in NSG mice and treatment with CD19, J591 and PSMA-VH T cells (n = 5 mice per group). (B) Representative images of tumor bioluminescence (BLI) at selected time points post T cell injections. (C) Kinetics of tumor BLI post T cell injections. (D) Schematic of the metastatic prostate cancer model using PC3-PSMA-FFIuc-eGFP tumor cells in NSG mice and treatment with low dose of CD19, J591 and PSMA-VH T cells (n 4 mice per group). (E) Representative images of tumor BLI at selected time points post T cell injections for low dose of T cells. (F) Kinetics of tumor BLI post T cell injections low dose of T cells. (G, H) Percentage of T cells in the gate of live cells (G) and total cell numbers (H) in blood, spleen, and bone marrow from PC3-PSMA-bearing mice treated with low doses of CAR- T cells. Mice were euthanized at day 58 after CAR-T cells infusion and T cells were identified as CD45 ⁺ CD3 ⁺ cells by flow cytometry. J594 group (n = 5) PSMA-VH group (n = 4). p >0.05 by t-test.

Figure 4. T cells expressing the human antibody VH domain-based CAR targeting MSLN demonstrate antitumor activity. (A) Schematic diagram of MSLN-scFv and MSLN-heavy- chain-only (MSLN-VH) CAR constructs. (B) Summary of coculture of CD19, MSLN.scFv and MSLN-VH T cells with Aspc-1-eGFP (MSLN ⁺ ) and PC3-eGFP (MSLN-) tumor cell lines. T cells were cocultured with tumor cells at an E:T ratio of 1 :5 for 6 days. At day 6, all cells were collected and analyzed by flow cytometry to quantify tumor cells and T cells, respectively. Error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (C) IFN-y (upper panel) and IL- 2 (lower panel) detected in the supernatants of the cocultures illustrated in (B) as measured by ELISA; error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (D) Representative flow cytometry plots showing the proliferation of MSLN.scFv and MSLN-VH T cells in response to tumor cells as assessed by CFSE dilution. Data are representative of 3 experiments. (E) Schematic of the metastatic pancreatic cancer model using Aspc-1-FFIuc- eGFP tumor cells in NSG mice. (F, G) Representative tumor BLI (F) and bioluminescence kinetics (G) of Aspc-1-FFIuc-eGFP tumor growth at the representative time points post T cell injections, (n = 5 mice per group). (H) Kaplan-Meier survival curve of mice in (E) (n= 5 mice per group). Data are representative of two experiments. (I) Frequency of human CD45 ⁺ CD3 ⁺ cells in blood at 22 days (left) post T-cell infusion and at euthanasia (right) of MSLN-scFv and MSLN-VH T cells, respectively. Data are shown as individual values and the mean (n = 5 mice per group), p >0.05 by t-test.

Figure 5. In vitro analysis of monospecific and bispecific Humabody V _H binding. (A) Schematic representation of monospecific (single VH) or bispecific (double VH) proteins. (B) Single cycle Biacore kinetic analysis of PSMA binding. (C) Biacore kinetic analysis of MSLN binding, 3-fold dilution series starting at 300nM, except the control scFv protein which started at 33.3nM. Data are representative of two experiments.

Figure 6. T cells expressing two human antibody VH domain-based CARs demonstrate dual specificity in vitro. (A) Schematic diagram of PSMA-VH, MSLN-VH, and PSMA/MSLN- VH CAR constructs. (B, C) Representative flow cytometry plots (B) and summary (C) illustrating CAR expression in T cells. The CD19-specific CAR (CD19) was used as negative controls. p >0.05 by One-way ANOVA. (D) Representative flow cytometry plots showing PC3-PSMA- eGFP (PSMA target), PC3-MSLN-eGFP (MSLN target), and mixture of PC3-PSMA-eGFP and PC3-MSLN-eGFP (1 :1 ratio) cotultured with CD19.CAR, PSMA-V _H .CAR, MSLN-VH. CAR and PSMA/MSLN-VH.CAR T cells at the E: T ratio of 1 :5 for 6 days. T umor cells and T cells were quantified at day 6 by flow cytometry. (E) Summary of coculture experiments illustrated in (D); error bars represent SD, (n = 5). *p < 0.05, **p < 0.01 , ****p < 0.0001 , Two-way ANOVA. (F, G) IFN-y (F) and IL-2 (G) detected in the coculture supernatant of the coculture experiments described in (D) as measured by ELISA; error bars represent SD, (n = 3) *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 , Two-way ANOVA. Figure 7. T cells expressing two human antibody VH domain-based CARs demonstrate dual specificity in vivo. (A) Schematic of the xenograft mouse model in which NSG mice were systemically engrafted with mixed FFIuc-eGFP labeled PC3-PSMA (5 x 10 ⁵ cells) and PC3-MSLN (5 x 10 ⁵ cells) cells at 1 :1 ratio, and treated with two doses of CAR-T cells at day 0 and day 7, respectively (6 x 10 ⁶ cells each dose, n = 5 mice per group). (B, C) Representative tumor BLI images (B) and bioluminescence kinetics (C) at selected time points post T cell injections. (D) Number of human CD45 ⁺ CD3 ⁺ cells in the peripheral blood collected at day 21 post second T-cell infusion in mice treated as described in (A). Data are shown as individual values and the mean (n = 5 mice per group) and are representative of two experiments, p >0.05 by one-way ANOVA. (E) Representative antigen expression pattern in the tumor cells isolated from the mice with relapsed tumor in mice treated as described in (A).

Figure 8. Heavy-chain-only-based CAR-T cells express LAG3 and PD-1 upon activation as scFv-based CAR-T cells. (A) Representative flow cytometry plots illustrating Granzyme- B expression in T cells expressing either J591 or PSMA-VH without co-culture with tumor cells (rest condition). (B-E) Representative flow plots and summary illustrating the kinetics of LAG3 (B,C) and PD-1 (D,E) expression in T cells expressing either J591 or PSMA-VH cocultured overnight with the tumor cell line expressing PSMA (PC3-PSMA-eGFP) at E:T ratio of 1 :2. Data are representative of 4 experiments. **p < 0.01 Two-way ANOVA.

Figure 9. Transduction efficiency of MSLN-scFv and MSLN-VH CARS. (A,B) Representative flow pots (A) and summary (B) illustrating MSLN-scfv and MSLN-VH expression in T cells. The CD19-specific CAR (CD19) and non-transduced T cells (NT) were used as positive and negative controls, respectively. ****p < 0.0001 , One-way ANOVA.

Figure 10. Expression of MSLN in Aspc-1, PC3 and engineered PC3 cells. Representative flow cytometry histograms showing the expression of MSLN in Aspc-1 , PC3 and PC3 cells engineered with retroviral vector to express MSLN. Figure 11. Bispecific heavy-chain-only-based CAR-T cells demonstrate dual specificity. (A) Representative flow cytometry plots showing coculture of PC3-PSMA-eGFP (PSMA target) and Aspc-1 -eGFP (MSLN target) tumor cells with CD19.CAR, PSMA-VH. CAR, MSLN-VH. CAR and PSMA-VH/MSLN-VH.CAR T cells at E:T ratio of 1 :5 for 6 days. At the end of co-culture, cells were collected to enumerate T cells (CD3) and tumor cells (GFP), respectively by flow cytometry. (B) Summary of coculture experiments illustrated in (A); error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA. (C,D) IFN-y (C) and IL-2 (D) released in the coculture supernatant of the experiments illustrated in (A) as measured by ELISA; error bars represent SD, (n = 4). ****p < 0.0001 , Two-way ANOVA.

Figure 12. Phenotypic characterization of T cells in the peripheral blood of mice in the PC3-PSMA and PC3-MSLN Mixed tumor model. CAR-T cells in the peripheral blood at day 21 post second T-cell infusion were identified by the expression of CD45 and CD3 by flow cytometry. PD1 (A), TIM3 (B), CD45RA and CCR7 expression (C) were examined, error bars represent SD, (n = 5). p >0.05 by One-way ANOVA. Figure 13: A)-O) Sequences of VH single domain antibodies that bind PSMA grouped according to family based on sequence similarity. This figure shows the full length VH sequence. CDR1 , CDR2 and CDR3 are highlighted in bold.

Detailed description

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2013)). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, immunology, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The invention relates to CAR constructs that include a human VH single domain antibody that specifically binds human MSLN and has the sequences described herein as well as to related products, methods and uses.

The term VH domain as used herein refers to an isolated single human VH domain antibody which is also termed VH sdAb. A VH domain is referred to as Humabody® herein. Humabody® is a registered trademark of Crescendo Biologies Ltd. These terms are thus used interchangeably.

The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to a single domain antibody that is substantially free of other single domain antibodies, antibodies or antibody fragments. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.

The terms “single domain antibody”, “single variable domain antibody”, “single variable heavy chain domain antibody”, “single VH domain antibody”, “immunoglobulin single variable domain (ISV)”, “immunoglobulin single variable domain antibody”, “VH single domain antibody”, “single heavy chain domain”, “single variable heavy chain domain”, “single VH domain” or “VH domain” are all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen.

A single variable “heavy chain domain antibody, single variable heavy chain domain, immunoglobulin single heavy chain variable domain (ISV), human VH single domain” etc as used herein therefore does not comprise any other parts of a full antibody, but only have the antigen binding VH domain; e.g. it only includes the VH domain and does not comprise constant heavy chain domains and does not comprise a light chain. A single variable heavy chain domain antibody is capable of specific binding to an antigen in the absence of light chain or other antibody fragments.

VH domains are small molecules of 12-14 kDa which can be combined into different formats (formatted Humabody®) to give multivalent or multispecific antigen binding domains of a CAR. VH domains are robust and are characterised by high affinity and stability in serum ³⁰ .

Each single VH domain antibody comprises three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Thus, in one embodiment of the invention, the domain is a human variable heavy chain (VH) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The term "CDR" refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The numbering system described by Kabat is used herein unless otherwise stated. Also, as used herein, the term VH or "variable domain" refers to immunoglobulin variable domains defined by Kabat et al.

The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (/.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et a/., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

As used herein, the VH domain is a human VH domain. The term “a human VH domain” includes a VH domain that is derived from or based on a human VH domain amino acid or nucleic acid sequence. Thus, the term includes variable heavy chain regions derived from human germline immunoglobulin sequences. The VH domain can be produced using known methods in the art.

For example, the term “human VH domain” includes VH domains that are isolated from transgenic mice expressing human immunoglobulin V genes, in particular in response to an immunisation with an antigen of interest, for example as described in WO 2016/062990. Such domains are preferably fully human. In one embodiment, a human VH domain can also include a VH domain that is derived from or based on a human VH domain amino acid or nucleic acid sequence encoding such VH domain. Thus, the term includes variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences. A substantially human VH domain or VH domain that is derived from or based on a human VH domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo). The term “human VH domain” therefore also includes a substantially human VH domain wherein one or more amino acid residue has been modified. For example, a substantially human VH domain the VH domain may include up to 10, for example 1 , 2, 3, 4 or 5 amino acid modifications compared to a fully human sequence.

However, the term "human VH domain" or "substantially human VH domain", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Preferably, the term "human VH domain", as used herein, is also not intended to include camelized VH domains, that is human VH domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the VH domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid VHH domain.

A VH domain is the smallest antigen binding fragment. The term "antibody" broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3,

CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , lgG2, IgG 3, lgG4, IgAI and lgA2) or subclass.

The term antibody fragment as used herein is a portion of an antibody, for example as F(ab')2, Fab (Fragment, antibody), scFv (single chain variable chain fragments), single domain antibodies (dAbs), Fv, sFv, and the like. Functional fragments of a full-length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments have been used to develop therapeutics as an alternative to therapeutics based on mAbs. scFv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domains are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

MSLN is a glycoprotein anchored to the plasma membrane by a glycophosphatidyl inositol (GPI) domain. It is initially synthesized as a 69 kDa cell-surface protein. After cleavage of the amino terminus by the furin protease, a 40-kDa C-terminal fragment remains attached to the membrane and a soluble 32-kDa N-terminal fragment, named MPF (megakaryocyte potentiating factor), is released. A soluble form of MSLN has also been detected in the sera of patients with solid tumors, which is referred to as soluble MSLN-related protein (SMRP) ²³ .

High mRNA expression of mesothelin is found in mesothelioma, lung, ovarian, breast and pancreatic adenocarcinomas. Mesothelin over-expression has also been noted in some other human cancers, including squamous cell carcinomas of different sites such as cervix, lung and head and neck carcinomas, endometrial adenocarcinomas, colorectal, gastric, and esophageal cancers ²³ .

As used herein, the terms mesothelin or MSLN refer to the 40-kDa protein, mesothelin, which is anchored at the cell membrane by a glycosylphosphatidyl inositol (GPI) linkage and its soluble form; e.g. a soluble MSLN that circulates in the serum of cancer patients. MSLN contain N-glycosylation sites. For example, the term refers to a human mesothelin of GenBank accession number AAH03512.1.

The human mesothelin protein sequence and nucleic acid sequences are shown below.

SEQ ID NO. 1 MSLN amino acid sequence

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPN ISSLS PRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDL LL FLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRA L GGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVST MDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPS GK KAREI DESLI FYKKWELEACVDAALLATQM DRVN Al PFTYEQLDVLKH KLDELYPQGYPESVI QHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKG R GQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARL AF QNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLG P HVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLG P GPVLTVLALLLASTLA

SEQ ID NO. 2 MSLN nucleic acid sequence

ATGGCCTTGCCAACGGCTCGACCCCTGTTGGGGTCCTGTGGGACCCCCGCCCTCGGC AGCCTCCTGTTCCTGCTCTTCAGCCTCGGATGGGTGCAGCCCTCGAGGACCCTGGCTG GAGAGACAGGGCAGGAGGCTGCGCCCCTGGACGGAGTCCTGGCCAACCCACCTAACA TTTCCAGCCTCTCCCCTCGCCAACTCCTTGGCTTCCCGTGTGCGGAGGTGTCCGGCCT GAGCACGGAGCGTGTCCGGGAGCTGGCTGTGGCCTTGGCACAGAAGAATGTCAAGCT CTCAACAGAGCAGCTGCGCTGTCTGGCTCACCGGCTCTCTGAGCCCCCCGAGGACCT GGACGCCCTCCCATTGGACCTGCTGCTATTCCTCAACCCAGATGCGTTCTCGGGGCCC CAGGCCTGCACCCGTTTCTTCTCCCGCATCACGAAGGCCAATGTGGACCTGCTCCCGA GGGGGGCTCCCGAGCGACAGCGGCTGCTGCCTGCGGCTCTGGCCTGCTGGGGTGTG

CGGGGGTCTCTGCTGAGCGAGGCTGATGTGCGGGCTCTGGGAGGCCTGGCTTGCGAC CTGCCTGGGCGCTTTGTGGCCGAGTCGGCCGAAGTGCTGCTACCCCGGCTGGTGAGC TGCCCGGGACCCCTGGACCAGGACCAGCAGGAGGCAGCCAGGGCGGCTCTGCAGGG CGGGGGACCCCCCTACGGCCCCCCGTCGACATGGTCTGTCTCCACGATGGACGCTCT GCGGGGCCTGCTGCCCGTGCTGGGCCAGCCCATCATCCGCAGCATCCCGCAGGGCAT CGTGGCCGCGTGGCGGCAACGCTCCTCTCGGGACCCATCCTGGCGGCAGCCTGAACG GACCATCCTCCGGCCGCGGTTCCGGCGGGAAGTGGAGAAGACAGCCTGTCCTTCAGG CAAGAAGGCCCGCGAGATAGACGAGAGCCTCATCTTCTACAAGAAGTGGGAGCTGGAA GCCTGCGTGGATGCGGCCCTGCTGGCCACCCAGATGGACCGCGTGAACGCCATCCCC TTCACCTACGAGCAGCTGGACGTCCTAAAGCATAAACTGGATGAGCTCTACCCACAAGG TTACCCCGAGTCTGTGATCCAGCACCTGGGCTACCTCTTCCTCAAGATGAGCCCTGAG GACATTCGCAAGTGGAATGTGACGTCCCTGGAGACCCTGAAGGCTTTGCTTGAAGTCA ACAAAGGGCACGAAATGAGTCCTCAGGCTCCTCGGCGGCCCCTCCCACAGGTGGCCA CCCTGATCGACCGCTTTGTGAAGGGAAGGGGCCAGCTAGACAAAGACACCCTAGACAC CCTGACCGCCTTCTACCCTGGGTACCTGTGCTCCCTCAGCCCCGAGGAGCTGAGCTCC GTGCCCCCCAGCAGCATCTGGGCGGTCAGGCCCCAGGACCTGGACACGTGTGACCCA AGGCAGCTGGACGTCCTCTATCCCAAGGCCCGCCTTGCTTTCCAGAACATGAACGGGT CCGAATACTTCGTGAAGATCCAGTCCTTCCTGGGTGGGGCCCCCACGGAGGATTTGAA GGCGCTCAGTCAGCAGAATGTGAGCATGGACTTGGCCACGTTCATGAAGCTGCGGACG

GATGCGGTGCTGCCGTTGACTGTGGCTGAGGTGCAGAAACTTCTGGGACCCCACGTG GAGGGCCTGAAGGCGGAGGAGCGGCACCGCCCGGTGCGGGACTGGATCCTACGGCA GCGGCAGGACGACCTGGACACGCTGGGGCTGGGGCTACAGGGCGGCATCCCCAACG GCTACCTGGTCCTAGACCTCAGCATGCAAGAGGCCCTCTCGGGGACGCCCTGCCTCCT AGGACCTGGACCTGTTCTCACCGTCCTGGCACTGCTCCTAGCCTCCACCCTGGCCTGA

Variants of the sequences above are also included. The term variant is defined elsewhere herein but includes biologically active variants with at least 90% sequence identity to the sequences shown above. Different isoforms of MSLN are also included. There are 4 different isoforms of mesothelin which are produced by alternative splicing. SEQ ID NO. 1 is isoform 1 which is the canonical sequence.

Isoforms 2, 3 and 4 differ from isoform 1 as follows:

Isoform 2: The sequence of this isoform differs from the canonical sequence as follows: residues 409-416: Missing.

Isoform 3: Also known as: SMRP. The sequence of this isoform differs from the canonical sequence as follows: residues 409-416: Missing. residues 601-630: MQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO. 508) VQGGRGGQARAGGRAGGVEVGALSHPSLCRGPLGDALPPRTWTCSHRPGTAPSLHPGL RAPLPC (SEQ ID NO. 509)

Isoform 4: The sequence of this isoform differs from the canonical sequence as follows: residues 44-44: Missing, residues 409-416: Missing.

The terms "MSLN binding molecule/protein/polypeptide/agent/moiety”, "MSLN antigen binding molecule molecule/protein/polypeptide/agent/moiety”, “anti- MSLN single domain antibody”, “anti- MSLN single immunoglobulin variable domain”, “anti- MSLN heavy chain only antibody” or “anti- MSLN antibody” all refer to a molecule capable of specifically binding to the human MSLN antigen. The binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity. Binding is to human MSLN unless otherwise defined.

A single domain antibody as described herein, "which binds" or is “capable of binding” an antigen of interest, e.g. human MSLN, is one that binds the antigen with sufficient affinity such that the CAR with the single domain antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen MSLN as described herein. Binding is to the extracellular domain of MSLN. In one embodiment, the invention relates to an isolated CAR comprising a VH single domain antibody that specifically binds to human MSLN wherein the VH single domain antibody comprises a CDR1 comprising or consisting of SEQ NO. 4 or a sequence with 1 , 2 or 3 amino acid modifications, a CDR2 comprising or consisting of SEQ NO. 5 or a sequence with 1 , 2, 3 or 4 amino acid modifications and a CDR3 comprising or consisting of SEQ NO. 6 or a sequence with 1 , 2 or 3 amino acid modifications.

SEQ ID NO. 3 VH single domain antibody full length amino acid sequence that binds mesothelin, also termed MSLN VH 1.1 (CDRs underlined):

EITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWND DKRYR PSLKNRLTIAKDTSKNQVVLTMTNMDPVDTARYYCAHYSTSSETAFDI RGQGTMVTVSS The CDRs of SEQ ID NO. 3 are as follows:

CDR1 SEQ ID NO. 4 : TSGLGVG

CDR2 SEQ ID NO. 5 : LIYWNDDKRYRPSLKN

CDR3 SEQ ID NO. 6 : YSTSSETAFDI

In one embodiment, the VH single domain antibody comprises or consists of SEQ ID NO. 3 or a variant thereof provided that the variant retains the CDRs as defined above. In one embodiment, the VH single domain antibody may be a variant of SEQ ID NO. 3 having one or more amino acid substitutions, deletions, insertions in the framework regions but retaining the CDRs as defined above.

In one embodiment, the variant (MSLN VH1.2) has a substitution of S to N in CDR3 and the CDR3 sequence is: SEQ ID NO. 7: YNTSSETAFDI. SEQ ID NO. 504 (Amino acid sequence of MSLN VH1.2):

EITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWND DKRYR PSLKNRLTIAKDTSKNQWLTMTNMDPVDTARYYCAHYNTSSETAFDIRGQGTMVTVSS

A variant as used herein retains a biological function of the single domain antibody, that is binding to the target antigen (e.g. MSLN) and, thus, the variant antibody or antibody fragment thereof can be sequence engineered. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence provided that the CDRs are as defined above.. Amino acid substitutions in variants as described herein can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Substitutions, insertions, additions or deletions in the framework region may optionally be in the range of about 1 to 25 or 1 to 50, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20 amino acids, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

In one embodiment, the modification (e.g. amino acid substitutions, insertion, addition or deletion) is in HCDR1 , HCDR2 and or HCDR3 as defined for the CDRs above.

In one embodiment, variations are only in the framework sequences and the VH single domain antibody CDR1 , 2 and 3 comprising or consisting of SEQ ID Nos 4, 5 and 6 respectively. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. As used herein, the term amino acid modification includes substitutions, additions and deletions. Modifications in the sequence of the single domain antibody can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e. antigen binding) using the functional assays described herein.

Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 1 below.

Table 1. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions

A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including in vitro and in vivo expression libraries. Yeast display technique are also included. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and I or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the antibodies described herein.

In one embodiment, modifications to the sequence can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence. More specifically, a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived. In one embodiment, all framework sequences are germline sequence.

To return one or more of the amino acid residues in the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.

In still another embodiment, glycosylation is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen.

The term variants describes a sequence variant with a different sequence (e.g. amino acid or nucleotide sequence) compared to a reference molecule and which retain biological activity. Variants can also be defined by reference to sequence identity.

In one embodiment, the one or more substitution is in the CDR1 , 2 or 3 region as follows: there may be 1 , 2, 3 or more amino acid substitutions in the CDR1 , 2 or 3. In another example, there may be 1 , 2, 3 amino acid deletions or addition.

In one embodiment, the one or more substitution, addition or deletion is in the framework region. For example, there may be 1 to 20, e.g. 1 to 10 or more amino acid substitutions in the framework regions.

Variants of the MSLN sequence shown herein may have at least 70%, 75%, 80%, 90% or 95% sequence identity to SEQ ID NO. 3 provided that the CDRs are as defined above.

Variants of other sequences (other than SEQ ID NO. 3) as shown herein may also have at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

In one embodiment, one or more non-germline residue in SEQ ID NO. 3 is replaced with a germline residue. Thus, the residue at position 1 , 34, 60, 65, 70 and/or 103 is replaced with the germline residue as shown below. In one embodiment, all of the residues at these positions are replaced with the germline residue.

Position (Kabat) residue in SEQ ID NO. 3 residue in germline (VH2-05)

1 E Q

34 L V

60 R S

65 N S

70 A T

103 R W As used herein, the terms sequence "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, for example with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST, FASTA, the Smith-Waterman algorithm, or the TBLASTN program. In particular, the psi-Blast algorithm may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments can be performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms.

“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g, antibody or antigen -binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).

Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. Kon refers to the association rate constant of, e.g, an antibody or antigenbinding fragment thereof to an antigen, and koff refers to the dissociation of, e.g, an antibody or antigen-binding fragment thereof from an antigen. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.

The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or lower. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The single VH domain antibodies used in the CAR of the invention may have KD, IC50 and/or EC50 values as further described herein and as shown in the examples. Suitable affinities may also be as described above.

In one embodiment, the VH affinity for MSLN used in the CAR as a single VH (monomer) recombinant protein is in the nanomolar range, e.g. 20 to 40 nM, e.g. 28-34nM. The VH affinity for MSLN used in the CAR as a bispecifics format is 10-60nM, e.g. 16-49nM. Measurement may be using BiaCore®.

The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody.

The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance), HTRF; flow cytometry; fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.

The inventors have surprisingly shown that a CAR with the VH single domain antibody as identified in SEQ ID NO. 3 has better tumour killing in vivo, despite the difference in affinity compared to a control scFv that binds MSLN (see examples and Fig. 4 and 5).

In one aspect the invention also relates to a VH single domain antibody that binds MSLN as described above in the use of a CAR.

Elements of the CAR

The terms "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refer to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the lytic capacity and self renewal of T cells. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. The term “antigen binding domain or “antigen-specific targeting domain" as used herein refers to the region of the CAR which targets and binds to specific antigens as explained above. When a CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain).

A skilled person would know that a CAR comprises additional elements. A skilled person would also know that such elements of a CAR (other than antigen-specific targeting domain described herein) are well known in the art. Thus, the invention is not limited to specific domains of the CAR in addition to the antigen-specific targeting domain described herein.

As mentioned above, the first generation CARs have been tested in various phase I clinical studies in patients with cancer. Second generation CARs and third generation CARs have also been described are more complex with three or more signalling domains (reviewed references 4 and 5 and in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009, Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11 , 69, 2021). CARs are also described in US2004043401 , W02019200007 and

WO2021108613, all incorporated herein by reference.

For example, the CAR of the invention may comprise a molecule of the general formula: MSLN binding human VH sdAb- transmembrane domain- Intracellular signaling domain. Exemplary domains are listed below. As will also be apparent, the CAR may comprise additional domains as explained below.

In one embodiment, the CAR may comprise a mesothelin binding VH single domain antibody as described herein, an extracellular domain (which may comprise a “hinge” domain), a transmembrane domain, and an intracellular signaling domain.

The Intracellular (Cytoplasmic) Domain The intracellular (cytoplasmic) domain of the CAR can provide activation of at least one of the normal effector functions of the immune cell. The CAR of the invention may thus further comprise an intracellular signaling domain. An "intracellular signaling domain", "cytoplasmic domain" or “endodomain” is the domain that transmits activation signals to T cells and directs the cell to perform its specialized function.

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

The intracellular domain may comprise at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof. The antigen binding molecule, i.e. the mesothelin binding VH single domain antibody may be engineered such that it is located in the extracellular portion of the molecule/construct, such that it is capable of recognizing and binding to its target or targets.

Examples of domains that transduce the effector function signal and can be used according to the invention include but are not limited to the chain of the T-cell receptor complex or any of its homologs (e.g., q chain, FcsRIy and chains, MB1 (Igalpha) chain, B29 (Igbeta) chain, human CD3zeta chain, CD3 gamma or other CD3 polypeptides (A, 5 and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5, 0X40 and CD28.

It will be appreciated that suitable intracellular molecules may also include but are not limited to, 4-1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-I, CDI-la/CDI8),

CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69,

SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In some embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.

The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBan Acc. No. BAG36664.1 , or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1.

The extracellular signaling domain

The CAR may also comprise an extracellular signaling domain. The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4-1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 , CDI-la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, IT GAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRAN CE/R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source. Hinge region

In one embodiment, the CAR of the invention further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. In particular, extracellular domains often comprise a hinge portion. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of the hinge or spacer region that can be used according to the invention include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer region will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In one embodiment, the hinge is an lgG4 hinge or a CD8A hinge, an immunoglobulin (Ig) sequence or other suitable molecule to achieve the desired special distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T, or the EC domain of CD28.

The transmembrane domain

The CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.

A "transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane and is connected to the endoplasmic signaling domain and the antigen binding domain, in case of the latter optionally via a hinge. In one embodiment, the transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In one embodiment, the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain.

In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4- 1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA- I, CDI-la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, lg alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM- 1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

In one embodiment, the CAR of the invention further comprises one or more co- stimulatory domains to enhance CAR-T cell activity after antigen specific engagement. Inclusion of this domain in the CAR of the invention enhances the proliferation, survival and/or development of memory cells. The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. The co-stimulatory domain is located intracellularly.

The co-stimulatory domain is a functional signaling domain obtained from a protein selected form the following group: CD28, CD137 (4-IBB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 (CD1 la/CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those of skill in the art. Multiple co- stimulatory domains can be included in a single CAR to recruit multiple signaling pathways. In one embodiment, the co-stimulatory domain is obtained from 4-1 BB. The term "4-1 BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2. In one embodiment, the term "4-1 BB costimulatory domain" refers to amino acid residues 214-255 of GenBank Acc. No.

AAA62478.2.

In one embodiment, the CAR of the invention further comprises a "linker domain" or "linker region" that connects different domains of the CAR. This domain includes an oligo- or polypeptide region from about 1 to 100 amino acids in length. Suitable linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain during cellular processing and localization of the CAR to the cellular membrane. In one embodiment, the leader sequence is a CD8A domain.

The CAR may further include a label, for example a label that facilitates imaging, such as a fluorescent label or other tag. This can, for example, be used in methods for imaging tumor binding. The label may be conjugated to the antigen binding domain.

Suitable detectable labels which may be conjugated to antibody molecules are known in the art and include radioisotopes such as iodine-125, iodine-131 , yttrium-90, indium-11 1 and technetium-99; fluorochromes, such as fluorescein, rhodamine, phycoerythrin, Texas Red and cyanine dye derivatives for example, Cy7 and Alexa750; chromogenic dyes, such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; phosphor or laser dyes with spectrally isolated absorption or emission characteristics; and chemical moieties, such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

The CARs described herein may be synthesized as single polypeptide chains. In this embodiment, the antigen-specific targeting regions are at the N- terminus, arranged in tandem and are separated by a linker peptide.

In one embodiment, the CAR comprises the CD8a hinge and transmembrane domain, the CD28 costimulatory domain and CD3 intracellular signaling domain ²⁴ . In one embodiment, the CAR comprises SEQ ID NO. 507 as shown below.

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSL VITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO. 507

Multispecific antigen binding domains

The inventors have shown that the VH domain single antibody that binds MSLN and is described herein can be used to generate bispecific CAR-T cells to simultaneously target two different targets, i.e. antigens (MSLN and a second target such prostate-specific membrane antigen (PSMA)) expressed by tumor cells, and therefore achieve better tumor control in solid tumors. The data in the examples show that such VH modules in bispecific format are capable of binding their specific target with the same affinity as their monovalent counterparts. The results also show that bispecific CARs generated by joining two human antibody VH domains can prevent tumor escape in tumor with heterogeneous antigen expression. Dual targeting using PSMA-VH/MSLN-VH-T cells controlled the tumor growth more effectively than either by single targeting.

Thus, the antigen binding domain used in a CAR of the invention may comprises a further antigen binding moiety, e.g. an antibody or antibody fragment thereof, in addition to the single VH domain antibody that binds MSLN as described herein. In one embodiment the further antigen binding moiety is single VH domain antibody. In one embodiment the further antigen binding moiety is single VH domain antibody that binds PSMA.

Thus, more than one VH domain, e.g. two or three VH single domain antibody, may be included.

In one embodiment, the antigen binding domain comprises or consists of a first VH single domain antibody which binds to MSLN and is as described above and a VH single domain antibody which binds to a second, that is different antigen. Thus, in certain embodiments, the invention relates to bispecific CARs.

The first target and the second target are not the same, i.e. are different targets, e.g., proteins; both may be present on a cell surface. Accordingly, a bispecific binding molecule as described herein can selectively and specifically bind to a cell that expresses (or displays on its cell surface) the first target (MSLN) and the second target (e.g. PSMA).

In another embodiment, the binding molecule comprises more than two antigen-binding domains providing a multispecific binding molecule. A multispecific antigen-binding domain as described herein can in addition to binding a first target (MSLN) bind one or more additional targets, i.e., a multispecific polypeptide can bind at least two, at least three, at least four, at least five, at least six, or more targets, wherein the multispecific polypeptide agent has at least two, at least, at least three, at least four, at least five, at least six, or more target binding sites respectively. The bispecific antigen-binding domain has the following formula: VH (A)- L-VH (B) wherein A or B is MSLN. V _H (A) is conjugated to V H (B), i.e. linked to VH (B), for example with a peptide linker. L denotes a linker for example a polypeptide linker.

Each VH comprises CDR and FR regions. Thus, the binding molecule may have the following formula: FR1(A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)-L-FR1 (B)-CDR1(B)- FR2(B)-CDR2(BA)-FR3(B)-CDR3(B)-FR4(B). The order of the single V _H domains A and B is not particularly limited, so that, within a polypeptide of the invention, single variable domain A may be located N-terminally and single variable domain B may be located C-terminally, or vice versa wherein A or B is MSLN. The term "peptide linker" refers to a peptide comprising one or more amino acids. A peptide linker comprises 1 to 44 amino acids, more particularly 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, linkers that include G and/or S residues, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n" is generally a number between 1 and 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the peptide is for example selected from GGGGS (SEQ ID NO: 8), GGGGSGGGGS (SEQ ID NO: 9), SGGGGSGGGG (SEQ ID NO: 10), GGGGSGGGGSGGGGS (SEQ ID NO: 11), GSGSGSGS (SEQ ID NO: 12), GGSGSGSG (SEQ ID NO: 13), GGSGSG (SEQ ID NO: 14), GGSG (SEQ ID NO: 15) and GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16). In one embodiment, the linker is (G4S)e or (G4S)s.

The addition antigen targeting moiety present which binds a second target and, together with the VH single domain antibody that targets MSLN as described herein, "binds" or is “capable of binding” an antigen of interest, i.e. targets, antigen with sufficient affinity such the CAR is useful in therapy in targeting a cell or tissue expressing the antigen.

As used herein, the term "target" refers to a biological molecule (e.g., antigen, peptide, polypeptide, protein, lipid, carbohydrate) to which a polypeptide domain which has a binding site can selectively bind. The target can be, for example, an intracellular target (such as an intracellular protein target) or a cell-surface target (such as a membrane protein, e.g., a receptor protein). Preferably, a target is a cell-surface target, such as a cell-surface protein.

In one embodiment, the additional target of the antigen binding domain of the CAR (i.e. in addition to binding to MSLN) is a tumor antigen. In one embodiment, the tumor antigen is associated with a hematologic malignancy. In another embodiment, the tumor antigen is associated with a solid tumor. In yet another embodiment, the tumor antigen is selected from the group consisting of PSMA, PSCA, BCMA, CS1 , GPC3, CSPG4, EGFR, CD123, 5T4, CD23, L1CAM, MUC16, R0R1 , SLAMF7, cKit, CD19, CD20, CD22, CD33, CD38, CD53, CD92, CD100, CD148, CD150, CD200, CD261 , CD262, CD362, R0R1 , CD33/IL3Ra, c-Met, Glycolipid F77, EGFRvlll, GD-2, NY-ESO-1 TCR or MAGE A3 TCR, human telomerase reverse transcriptase (hTERT), survivin, cytochrome P450 1 B1 (CY1 B), HER2, Wilm’s tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16, MLIC1 , p53, cyclin, an immuno checkpoint target (e.g. PD-1 , LAG-3, TIM-3) or combinations thereof. However, a skilled person would understand that other tumor antigens are also targets within the scope of the invention.

In one embodiment, the antigen binding domain of the CAR comprises binds VH single domain antibody that targets MSLN as described herein and also comprises an antibody or antibody fragment that binds PSMA. In one embodiment, the antibody fragment is a VH single domain antibody that binds specifically PSMA.

Binding to PSMA is to wild type human PSMA (accession NO. Q04609). The sequence for the wild type human PSMA monomer is shown below (SEQ ID NO. 17).

1 MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEAT NITPKHNMKA 61 FLDELKAENI KKFLYNFTQI PHLAGTEQNF QLAKQIQSQW KEFGLDSVEL AHYDVLLSYP 121 NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDIVPP FSAFSPQGMP EGDLVYVNYA 181 RTEDFFKLER DMKINCSGKI VIARYGKVFR GNKVKNAQLA GAKGVILYSD PADYFAPGVK

241 SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGIAEAVGLP SIPVHPIGY 301 DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTN EVTRIYNVIG 361 TLRGAVEPDR YVILGGHRDS VWFGGIDPQS GAAVVHEIVR SFGTLKKEGW RPRRTILFAS 421 WDAEEFGLLG STEWAEENSR LLQERGVAYI NADSSIEGNY TLRVDCTPLM YSLVHNLTKE 481 LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGI ASGRARYTKN 541 WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANS IVLPFDCRDY 601 AVVLRKYADK IYSISMKHPQ EMKTYSVSFD SLFSAVKNFT EIASKFSERL QDFDKSNPIV 661 LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGIYD ALFDIESKVD 721 PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA

In one embodiment, the antigen binding domain includes a VH single domain antibody that bind PSMA which with the following sequence or a variant thereof.

SEQ ID NO. 18 (termed clone 2.1) full length sequence

EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSN KY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ GTMVTVSS

The CDRs of SEQ ID NO. 18 are shown below CDR1 SEQ ID NO. 19 : GYGMH

CDR2 SEQ ID NO. 20 : YISYDGSNKYYADSVKG

CDR3 SEQ ID NO. 21 : DPAWGLRLGESSSYDFDI

Suitable VH single domain antibodies that bind PSMA are described in WO 2017/122017 and WO2019/012260, incorporated herein by reference.

The variant may have one or more amino acid modification, that is a substitution, deletion or addition. In one embodiment, the VH single domain antibody comprises a CDR1 comprising SEQ NO. 19 or a sequence with 1 , 2 or 3 amino acid modifications, a CDR2 comprising SEQ NO. 20 or a sequence with 1 , 2 or 3 amino acid modifications and a CDR3 comprising SEQ NO. 21 or a sequence with 1 , 2 or 3 amino acid modifications.

In one embodiment, the one or more amino acid modification n is in the CDR1 , 2 or 3 region. For example, there may be 1 , 2, 3 or more amino acid modification in the CDR1 , 2 or 3.

In one embodiment, the one or more substitution, addition or deletion is in the framework region. For example, there may be 1 to 20, e.g. 1 to 10 or more amino acid substitutions framework regions.

In one embodiment, the VH single domain antibody that binds PSMA has at least 60%, 70%, 80% or 90% homology or identity to SEQ ID NO. 18, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to SEQ ID NO. 18. In one embodiment, said sequence homology or identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%,

84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, the VH single domain antibody that binds PSMA is selected from one of the VH single domain antibody as shown in Table 2b shown below. These molecules are related in sequence to SEQ ID NO. 18.

In one embodiment, the VH single domain antibody that binds PSMA is selected from one of the VH single domain antibody as shown in Table 2 shown below.

Table 2a Family 1 PSMA binders

Table 2b Family 2 PSMA binders. Note that the PSMA binder identified in SEQ ID 18 belongs to this family.

Table 2c Family 3 PSMA binders

Table 2d Family 4 PSMA binders

Table 2e Family 5 PSMA binders

Table 2f Family 6 PSMA binders

Table 2g Family 7 PSMA binders

Table 2h Family 8 PSMA binders

Table 2i Family 9 PSMA binders Table 2j Family 10 PSMA binders

Table 2kFamily 11 PSMA binders

Table 2I Family 12 PSMA binders

Table 2m Family 13 PSMA binders

Table 2n Family 14 PSMA binders

Table 2o Family 15 PSMA binders

In another embodiment, the VH single domain antibody that binds PSMA is selected from one of the VH single domain antibody as shown in figure 13, i.e. SEQ ID NO. 24 to 407 as shown above.

A linker such as polypeptide linker (e.g. (G4S)n) may be used to link the VH single domain antibody that binds MSLN with the VH single domain antibody that binds PSMA. The PSMA binder may be located N or C terminally. In one embodiment, the PSMA binder is a VH single domain antibody and is located N terminally. In one embodiment, the PSMA binder is a VH single domain antibody and is located N terminally and the linker is selected from (G4S)e or (G4S)3-

In one embodiment, the bispecific antigen binding domain used in the CAR has a polypeptide sequence selected from SEQ ID NO. 505 or 506.

The affinity of the monospecific antigen binding domain used in the CAR to huPSMA may be in the picomolar range, e.g. about 50-500 pM. Measurement may be using BiaCore.

The affinity of bispecific antigen binding domain used in the CAR to huPSMA may be in the picomolar range, e.g. about 100 to about 250pm, for example 116-213pM. Measurement may be using BiaCore.

Exemplary methods for making the VH single domain antibody

In one embodiment, the VH single domain antibody for use in a CAR as described herein is generated from human heavy chain only antibody produced in a transgenic rodent that expresses human heavy chain loci. The transgenic rodent, for example a mouse, may have a reduced capacity to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes. The rodent may therefore comprise modifications to disrupt expression of endogenous light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced.

For example, the rodent is a mouse or a rat. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, the mouse does not make a functional endogenous lambda light chain. In one embodiment, the lambda light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. For example, at least the constant region genes C1 , C2 and C3 may be deleted or rendered non-functional through insertion or other modification as described above. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional lambda light chain.

Furthermore, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, the mouse does not make a functional endogenous kappa light chain. In one embodiment, the kappa light chain locus is deleted in part or completely or rendered nonfunctional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional kappa light chain.

The mouse having functionally silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO 2003/000737, which is hereby incorporated by reference in its entirety. Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus. Thus, the mouse does not make a functional endogenous heavy chain. In one embodiment, the heavy chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional heavy chain.

For example, as described in WO 2004/076618 (hereby incorporated by reference in its entirety), all 8 endogenous heavy chain constant region immunoglobulin genes (p, 5, y3, y1 , y2a, y2b, s and a) are absent in the mouse, or partially absent to the extent that they are nonfunctional, or genes 5, y3, y1 , y2a, y2b and s are absent and the flanking genes p and a are partially absent to the extent that they are rendered non-functional, or genes p, 5, y3, y1 , y2a, y2b and s are absent and a is partially absent to the extent that it is rendered non-functional, or 5, y3, y1 , y2a, y2b, s and a are absent and p is partially absent to the extent that it is rendered non-functional. By deletion in part is meant that the endogenous locus gene sequence has been deleted or disrupted, for example by an insertion, to the extent that no functional endogenous gene product is encoded by the locus, i.e., that no functional product is expressed from the locus. In another embodiment, the locus is functionally silenced. For example, the mouse comprises a non-functional endogenous heavy chain locus, a nonfunctional endogenous lambda light chain locus and a non-functional endogenous kappa light chain locus. The mouse therefore does not produce any functional endogenous light or heavy chains. Thus, the mouse is a triple knockout (TKO) mouse.

The transgenic mouse may comprise a vector, for example a Yeast Artificial Chromosome (YAC) for expressing a heterologous heavy chain locus. YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells.

For example, the YAC may comprise multiple human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions.

Alternative methods known in the art may be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions.

Transgenic mice can be created according to standard techniques as illustrated in the examples. The two most characterised routes for creating transgenic mice are via pronuclear microinjection of genetic material into freshly fertilised oocytes or via the introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos. Regardless of how the genetic material is introduced, the manipulated embryos are transferred to pseudopregnant female recipients where pregnancy continues and candidate transgenic pups are born.

The main differences between these broad methods are that ES clones can be screened extensively before their use to create a transgenic animal. In contrast, pronuclear microinjection relies on the genetic material integrating to the host genome after its introduction and, generally speaking, the successful incorporation of the transgene cannot be confirmed until after pups are born.

There are many methods known in the art to both assist with and determine whether successful integration of transgenes occurs. Transgenic animals can be generated by multiple means including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are various tools and techniques that can be used to both drive and select for transgene integration and subsequent modification including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques, and nucleases to improve the efficiency of recombination. Most of these methods are commonly used in the modification of ES cells. However, some of the techniques may have utility for enhancing transgenesis mediated via pronuclear injection.

Further refinements can be used to give more efficient generation of the transgenic line within the desired background. As described above, in preferred embodiments, the endogenous mouse immunoglobulin expression is silenced to permit sole use of the introduced transgene for the expression of the heavy-chain only repertoire that can be exploited for drug discovery. Genetically manipulated mice, for example TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain and mouse lambda chain) can be used as described above. The transfer of any introduced transgene to this TKO background can be achieved via breeding, (either conventional or with the inclusion of an IVF step to give efficient scaling of the process). However, it is also possible to include the TKO background during the transgenesis procedure. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for use in transgenesis. Triple knock-out mice into which transgenes have been introduced are referred to herein as TKO/Tg. In one embodiment, the mouse is as described in WO 2016/062990.

The transgenic rodent described above produces human variable heavy chains which can be isolated and used for the generation of human VH domains, for example as described in WO 2016/062990, wO2016/113556, reference 30.

Polynucleotides, cells and methods for generating cells

In another aspect, the invention relates to an isolated nucleic acid molecule or construct comprising encoding a CAR as defined above. Such construct comprises the nucleic acid encoding a VH domain that targets MSLN as described herein (including variants) and additional nucleic acids which encode elements of the CAR. Exemplary nucleic acids are shown below and variant thereof, e.g. with 1 to 10 amino acid modifications, are also included. The nucleic acid sequence encoding SEQ ID NO. 3 may be as shown below or a variant thereof. GAGATCACCT TGAAGGAGTC TGGTCCTACG CTGGTGAAAC CCACACAGAC CCTCAC GCTG ACCTGTACCT TCTCTGGCTT CTCACTCAGC ACTAGTGGAC TGGGTGTGGG CT GGATCCGT CAGCCCCCAG GAAAGGCCCT AGAATGGCTT GCACTCATTT ATTGGAAT GA TGATAAACGC TACAGACCAT CTCTGAAGAA CAGGCTCACC ATCGCCAAGG ACAC CTCCAA AAACCAGGTG GTCCTTACAA TGACCAACAT GGACCCTGTG GACACAGCCA GATATTACTG TGCACATTAT AGCACCTCGT CCGAGACTGC TTTTGATATC CGGGGCC AAG GGACAATGGT CACCGTCTCC TCA SEQ ID NO. 22

In one embodiment, the variant has a substitution and the C underlined above at position 117 is replaced with an A.

The nucleic acid sequence encoding SEQ ID NO. 18 may be as shown below or a variant thereof.

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGGAAGTAATA AATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC TGGGGCCAAGGGACAATGGTCACCGTCTCCTCA

SEQ ID NO. 23

Nucleic acids encoding other PSMA VH domains as described herein can also be included. For example, the nucleic acid may encode one of the VH amino acid sequences shown in Table 2. Exemplary sequences are listed below.

Family 1

SEQ ID NO. 408 (encodes VH domain 1.1)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCATGAGTTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATGATGGTACCACA GACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAGTAT GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 409 (encodes VH domain 1.2)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATAATAATAATAGCACA GAGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCA CGCTGTATCTGCAAATGAACAGCCTGAGCGCCGAGGACACGGCCGTATATTACTGTGT GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA

SEQ ID NO. 410 (encodes VH domain 1.3)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCTCCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA GGCTCCAGGGAAGGGACTGGAGTGGGTCTCAAGTATTGGTGATAATAATAATAGCACA GACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGTA

CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

SEQ ID NO. 411 (encodes VH domain 1 .4)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATGGAACCACATACTA C

GCAGACTCCGTGAAGGGCCGTTTCACCATCTCCAGAGACAATTCCAAGAGCACGCTG T

ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAG A TGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

SEQ ID NO. 412 (encodes VH domain 1 .5)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCACTTATGCCATGAGCTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAAAATGATCGAACCAC A

TACTACGTAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGC A

CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTG C

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACTGTCTCTTCA

SEQ ID NO. 413 (encodes VH domain 1.6)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATAATAATAGAACCAC A

TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGC A

CGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTG C

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 414 (encodes VH domain 1 .7)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGATGGAACCACATACTA C

GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAGCACGCTG T ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGA

TGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 415 (encodes VH domain 1.8) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCATGAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATGATGGTACCAC A

GACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC

GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGT G

AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 416 (encodes VH domain 1 .9)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCACA

GACTACGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC

GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGT G

AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 417 (encodes VH domain 1.10)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCAC A

GACTACGCAGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC

GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGT G AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 418 (encodes VH domain 1.11)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCAC A

GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 419 (encodes VH domain 1.12)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCAC A

GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO. 420 (encodes VH domain 1.13)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCAC A

GACTACGCAGCCGACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 421 (encodes VH domain 1.14)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGCCA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCAC A

GACTACGCAGACGTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 422 (encodes VH domain 1.15)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCAC A

GACTACGCAGCCTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGTG

AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 423 (encodes VH domain 1.16)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCAC A

GACTACGCAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 424 (encodes VH domain 1.17) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCAC A

GACTACGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGT

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 425 (encodes VH domain 1.18)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGCTACCAC A

GACTACGCAGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO. 426 (encodes VH domain 1.19)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACGATACCAC A

GACTACGCAGCCTACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC

GCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTGT G

AAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 427 (encodes VH domain 1.20)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAGTTTTAGCAGCTATGCCCTCAGTTGGGTCCGC CA GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGGTGAGAATAACCATACCACA

GACTACGCAGCCACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT A

CGCTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCCGTCTATTACTGTG T

GAAAGATGGTGTCCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO. 428 (encodes VH domain 2.3)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCAGCTTCAGTGGCTATGGCATGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACATATATCATATGATGGAAGTAAT A

GATACTATGCAGAATCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAATTCCAAGA AC

ACGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGACATT

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 429 (encodes VH domain 2.4) CAGGTCACCTTGAAGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAAA

CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC C

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAGAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGAAAT CT

GGGGCCAAGGGACAATGGTCACCGTCTCCTCA

SEQ ID NO. 430 (encodes VH domain 2.5)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGCC

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CACTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTTATCGTCCTATGATTTTGAAAT TT

GGGGCCAAGGGACAATGGTCACCGTCTCTTCA

SEQ ID NO. 431 (encodes VH domain 2.6)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC C

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA A ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA

CGCTATATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAACTATCGTCCTATAAATTTGAAAT CT

GGGGCCAAGGGACAATGGTCACCGTCTCTTCA

SEQ ID NO. 432 (encodes VH domain 2.7)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGTAAT A

AATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGATATC

TGGGGCCAAGGGACAATGGTCACCGTCTCCTCA

SEQ ID NO. 433 (encodes VH domain 2.8)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC C AGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCAGTTATATCATATGATGGAAGTAATAA

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGAAAT CT

GGGGCCAAGGTACAATGGTCACCGTCTCCTCA

SEQ ID NO. 434 (encodes VH domain 2.9)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC C

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA A ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA

CGCTGTATCTGCAAATGAACAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGCAATCGTCCTATGCTTTTGAAAT CC

GGGGCCAGGGGACAACGGTCACCGTCTCTTCA

SEQ ID NO. 435 (encodes VH domain 2.10)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTGGCTATGGCATGCACTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATATATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA GA

CGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCATATGATTTTGATATCT

GGGGCCAAGGGACAATGGTCACCGTCTCCTCA

SEQ ID NO. 436 (encodes VH domain 2.11)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAAT A

AATACTATGCACCCAGCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACCGTCTCCTCA SEQ ID NO. 437 (encodes VH domain 2.12)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAAT AA

ATACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATAT CT

GGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 438 (encodes VH domain 2.13)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAAT A

AATACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 439 (encodes VH domain 2.14)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCGTGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAAT A

AATACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA SEQ ID NO. 440 (encodes VH domain 2.15)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAAT AA

ATACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATAT CT

GGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 441 (encodes VH domain 2.16)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCGCGCACTGGGTCCGC

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATAAGAGTAAT AA

ATACTATGCAGACAAGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATATCT

GGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 442 (encodes VH domain 2.17)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAAT A

AATACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 443 (encodes VH domain 2.18)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCAGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAAT A

AATACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 444 (encodes VH domain 2.19) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCTTCCACTGGGTCCG CC

AGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGATGCGAGTAATA A

ATACTATGCAGACAACGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATAT CT

GGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 445 (encodes VH domain 2.20)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCAGCTTCAGTGGCTATGGCATGCACTGGGTCCG C CAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAGTAATA

GATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTCTCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGATCCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGAAA TT

TGGGGCCAAGGGACAATGGTCACCGTCTCCTCA SEQ ID NO. 446 (encodes VH domain 2.21)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAAA

CTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCGC C

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTCTCTACAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG C

GAAAGATCCGGCCTGGGGATTACGTTTGGGGAAATTATCGTCCTATGATTTTGAAAT CT

GGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 447 (encodes VH domain 2.22) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCACGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGGGAGTAAT A

AATACTATGCAGCCCCGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGACGCGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 448 (encodes VH domain 2.23)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCACGCACTGGGTCCG C CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAATA

AATACTATGCATCCAGCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC

ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGT G

CGAAAGACCGGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 449 (encodes VH domain 2.24)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCATGCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACGAGAGTAAT A

AATACTATGCAAGGCTGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA AC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTG

CGAAAGACACGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGATA TC

TGGGGCCAAGGGACAATGGTCACTGTCTCTTCA

SEQ ID NO. 450 (encodes VH domain 2.25) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTGGCTATGGCCTCCACTGGGTCCG C

CAGGCTCCAGGCAAGGGACTGGAGTGGGTGGCATATATATCATATGACCTGAGTAAT A

AATACTATGCAAGGGGGGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGA A

CACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG T

GCGAAAGACGTGGCCTGGGGATTACGTTTGGGGGAGTCATCGTCCTATGATTTTGAT AT

CTGGGGCCAAGGGACAATGGTCACTGTCTCCTCA family 3

SEQ ID NO. 451 (encodes VH domain 3.1) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGACATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGAGATGAGGACACGGCTCTATATTACTGTG CG

AGAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCTTCA

SEQ ID NO. 452 (encodes VH domain 3.2)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATATCATATGATGGAAGTAATAAA

TATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AAAGATCGTATAGTGGGAGCCAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCCTCA

SEQ ID NO. 453 (encodes VH domain 3.3)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCCTCATTAGCTATGGCATGAACTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA CGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGC

GAAAGATCGTATAGTGGGAGCTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGG G

ACAATGGTCACCGTCTCCTCA

SEQ ID NO. 454 (encodes VH domain 3.4) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGCGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATA G

ATATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTG CG AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA CAATGGTCACCGTCTCCTCA

SEQ ID NO. 455 (encodes VH domain 3.5)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC AATGGTCACCGTCTCCTCA

SEQ ID NO. 456 (encodes VH domain 3.6)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC

GCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA

AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA C AATGGTCACCGTCTCCTCA

SEQ ID NO. 457 (encodes VH domain 3.7)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTCATCTGCAAATGGACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC

AATGGTCACTGTCTCTTCA

SEQ ID NO. 458 (encodes VH domain 3.8)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAGA

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAGGGAAC CCTGGTCACTGTCTCCTCA

SEQ ID NO. 459 (encodes VH domain 3.9)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATATCATATGATGGAAGTAATAG A TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC

GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA

AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA C AATGGTCACCGTCTCTTCA

SEQ ID NO. 460 (encodes VH domain 3.10)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC

AATGGTCACTGTCTCCTCA

SEQ ID NO. 461 (encodes VH domain 3.11)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTG CG AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA CAATGGTCACTGTCTCCTCA SEQ ID NO. 462 (encodes VH domain 3.12)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATA G ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA CGCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCG

AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCCTCA

SEQ ID NO. 463 (encodes VH domain 3.13)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC

AATGGTCACTGTCTCCTCA

SEQ ID NO. 464 (encodes VH domain 3.14)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTG CG AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA CAATGGTCACTGTCTCCTCA SEQ ID NO. 465 (encodes VH domain 3.15)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC AATGGTCACCGTCTCCTCA

SEQ ID NO. 466 (encodes VH domain 3.16)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCCA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTCATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGC GA AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC

AATGGTCACCGTCTCCTCA

SEQ ID NO. 467 (encodes VH domain 3.17)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCTGTATATTACTGTGC GA

AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA C AATGGTCACCGTCTCCTCA

SEQ ID NO. 468 (encodes VH domain 3.18)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGACTGGGTGGCATTTATAACATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTTTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCTGTATATTACTGTG CG

AAAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCCTCA

SEQ ID NO. 469 (encodes VH domain 3.19) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGACATATGATGGAAGTAATAG A

TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AGAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCTTCA

SEQ ID NO. 470 (encodes VH domain 3.20)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGC CA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGGCAGTAATAGA

TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AGAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A

CAATGGTCACCGTCTCTTCA SEQ ID NO. 471 (encodes VH domain 3.21)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGGCAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AGAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A CAATGGTCACCGTCTCTTCA

SEQ ID NO. 472 (encodes VH domain 3.22) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTCAGACATATGATGCCAGTAATAG A

TACTATGCAGACGCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AGAGATCGTATAGTGGGAGGTAGGGTCCCTGATGCTTTTGATATCTGGGGCCAAGGG A CAATGGTCACCGTCTCTTCA

SEQ ID NO. 473 (encodes VH domain 3.23)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGC CA GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATAACATATGATGGAAGTAATAGA

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGC GA

AAGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGA C AATGGTCACTGTCTCCTCA

SEQ ID NO. 474 (encodes VH domain 3.24)

AGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGAC

TCTCCTGTGCAGCCTCTGGATTCCCCTTAATTAGCTATGGCATGAATTGGGTCCGCC AG

GCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATAACATATGATGGAAGTAATAGA T

ACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA CG CTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAA

AGATCGTATTGTGGGAGCTAGGGTCCCTGATGCTTATGATATCTGGGGCCAAGGGAC A ATGGTCACTGTCTCCTCA family 4

SEQ ID NO. 475 (encodes VH domain 4.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCTTGAGA

CTCTCCTGTGTAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCGGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTATTGTGC GA

AAGAGAGGATTTTTGGAGTGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGGA CA

ACGGTCACCGTCTCCTCA

SEQ ID NO. 476 (encodes VH domain 4.2)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGCCA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAG A

TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC

GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTGC G

AAAGAGAGGATTTTTGGAGTGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGG AC

AACGGTCACTGTCTCCTCA

SEQ ID NO. 477 (encodes VH domain 4.3)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCCCCTTCATTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAGCTAATAG A TACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAC

GCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTATTGTGC GA

AAGAGAGGATTTTTGGCGTGCTTACCCCTGATGATTTTGAAATCTGGGGCCAAGGGA CA

ACGGTCACCGTCTCCTCA

SEQ ID NO. 478 (encodes VH domain 4.4)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTCACTAGCTATGGCATGCACTGGGTCCG CC

AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATA G

ATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTG C GAAAGAGAGGATTTTTGGAGCGCTTACCCCTGATGATTTTGATATCTGGGGCCAAGGGA

CAACGGTCACCGTCTCTTCA family 5

SEQ ID NO. 479 (encodes VH domain 5.1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAATACTAA AT

ATTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA CG

CTGTATCTGCAAATGAATAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCG AA AGGTTTATGGCCTTCGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCTTCA

SEQ ID NO. 480 (encodes VH domain 5.2)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACTATGGCATGCACTGGGTCCG CC AGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAATTATATCATATGATGGAAATAGTAA

ATATTATACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAA CA

CGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTG CG AAAGGTTTATGGCCTTCGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA family 6

SEQ ID NO. 481 (encodes VH domain 6.1)

CAGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG GT

CCGCCAGCACCCAGGGAAGGACCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAG AA CCAGTTCTCCCTGAAAATGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTG

CGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA

SEQ ID NO. 482 (encodes VH domain 6.2)

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG AT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAG AA

CCAGTTCTCCCTGAAAATGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG T

GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

SEQ ID NO. 483 (encodes VH domain 6.3)

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG GT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAGAA

CCAGTTCTCCCTGAAACTGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG T

GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

SEQ ID NO. 484 (encodes VH domain 6.4)

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG AT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTATCATATCAGTAGACACGTCTAAG AA CCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGT

GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

SEQ ID NO. 485 (encodes VH domain 6.5)

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG GT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAG AA

CCAGTTCTCCCTGAAAATGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG T

GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

SEQ ID NO. 486 (encodes VH domain 6.6)

CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG GT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAG AA

CCAGTTCTCCCTGAAACTGAACTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG T

GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

SEQ ID NO. 487 (encodes VH domain 6.7) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC

CTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAATAGTGGTTATTACTGGAGCTGG GT

CCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTTCATCTATTACAATGGGAG C

ATCCACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAG AA

CCAGTTCTCCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTG T GCGAGAGACGGGGATGACTACGGTGACTACTTGAGGGGCCAGGGAACCCTGGTCACC

GTCTCCTCA family 7

SEQ ID NO. 488 (encodes VH domain 7.1)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA

SEQ ID NO. 489 (encodes VH domain 7.2)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C

GAGAGATAACCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA SEQ ID NO. 490 (encodes VH domain 7.3)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGGGGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACT

SEQ ID NO. 491 (encodes VH domain 7.4)

GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGCCA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACCAGGGAAGTGAGAA ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACT CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCGC GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC

CGTCTCCTCA

SEQ ID NO. 492 (encodes VH domain 7.5)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACCCCGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C

GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA

SEQ ID NO. 493 (encodes VH domain 7.6)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGAGGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C

GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA

SEQ ID NO. 494 (encodes VH domain 7.7) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA

GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACATCGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C

GAGAGATTCCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA

SEQ ID NO. 495 (encodes VH domain 7.8)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGTACTGGGTCCGC CA GGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAATCACGATGGAAGTGAGAA

ATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CT

CACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGCG C

GAGAGATACCCTTATAGTGGGAGAGAGGGGCTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA family 8

SEQ ID NO. 496

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCC

CTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGC C

AGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCA

ACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACC AG

TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCG A

GAGGCCCCATACCAGCCACTGCTATACCCGATGCTTTTGATATCTGGGGCCAAGGGA C AATGGTCACTGTCTCCTCA family 9

SEQ ID NO. 497

GAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCC

CTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTCACTACTGGAGCTGGATCCGC C

AGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGACATAAATCATAGTGGAAGCACCA

ACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAATC AG

TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGTG A

GAGACTACGGTGACTCCCGTAGCCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCA C CGTCTCTTCA family 10 SEQ ID NO. 498

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCATTTATGTCATATGATGGCAGTAATAA A

TACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC

GCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC G

AAAGGCGATTACGATTTTTGGAGTGGTTACCCCGACTACGATATGGACGTCTGGGGC C

AAGGGACCACGGTCACCGTCTCCTCA family 11

SEQ ID NO. 499 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCAACTTGATTAGCTATGGCATGTACTGGGTCCGC CA

GGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAA A

AACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAT AC GCTGTTTCTGCAAATGAACAGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGA

AAGGGGGGAATGCCTTGTATAGCAGTGGCTGGCCCGATGATGGTTTTGATATCAGGG G

CCAAGGGACAATGGTCACTGTCTCCTCA family 12

SEQ ID NO. 500

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGA

CTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTTTGGCATGCACTGGGCCCGC CA

GGCTCCAGGCAAGGGACTGGAGTGGGTGGCAGTAATATCATATGATGGAAATAGTAA A

TACTATGCAGACACCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC AC GCTGTATCTGGAAATGAACAGCCTGAGAGCTGATGACACGGCTGTGTATTACTGTGCGA

AAGGCCTATGGCCCCCAATGGACGTCAGGGGCCAAGGGACCACGGTCACCGTCTCCT CA family 13

SEQ ID NO. 501

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTCCAGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTGACTATTGGATGACCTGGGTCCG CC

AGGTTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGA

AATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGA AC

TCACTATATCTGCAAATGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGT GC GAGAGATCGAGGAGGAGCAGTGGCCCTTTATCACAACGGTATGGACATGGGGGGCCA AGGGACCACGGTCACTGTCTCTTCA family 14

SEQ ID NO. 502

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT

CTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACA GG

CCACTGGACAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGGTAACACAG G

CTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCAC A

GCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCG A

GAGGGAACGGGCCCGGTATAACTGGAACTACTGACTACTGGGGCCAGGGAACCCTGG TCACTGTCTCTTCA family 15

SEQ ID NO. 503 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCC AAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATCGTAC CGGTTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC TCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGG GAGAGAGAATGTTATAGTACCAGCTGCTACCTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA

The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA. RNA includes in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein. The nucleic acid may further comprise a suicide gene. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

In another aspect, the invention relates to an isolated nucleic acid construct comprising a nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases.

In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses such as adenovirus vectors can be used. In one embodiment, a lentivirus vector is used. The retrovial SFG vector is demonstrated in the examples.

In a further aspect, the invention also relates to an isolated cell or cell population comprising one or more nucleic acid construct or vector as described above. In one embodiment, the cell is an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, plant, mammalian or other suitable host cell.

Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae,

Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.

The invention also provides an isolated and genetically engineered cell or cell population which comprises and stably express a CAR nucleic acid construct or vector of the invention. Thus, the cell or cell population comprises the nucleic acid molecule encoding a CAR molecule having an antigen binding domain as described herein and further optional domains as described herein, e.g. an intracellular domain, a transmembrane domain and an extracellular domain.

In one embodiment, the cell is an immune cell. In one embodiment, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR-expressing cell, dendritic cell, or NK-T cell and a regulatory T cell, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells. T cells may be isolated from a patient for transfection with a CAR nucleic acid construct of the invention.

In one embodiment, the cell is an autologous T cell or allogeneic T cell.

In some embodiments, a source of cells, e.g., T cells or natural killer (NK) cells, can be obtained from a subject, e.g. a human patient. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.

The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. The cells may be washed with PBS. As will be appreciated, a washing step may be used, such as by using a semiautomated flowthrough centrifuge, for example, the Cobe™ 2991 cell processor, the Baxter CytoMate™, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solution with or without buffer. In certain embodiments, the undesired components of the apheresis sample may be removed.

In certain embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28 ⁺ CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and CD45RO ⁺ T cells can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4<+>cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, GDI lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention. PBMCs may be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.

In some embodiments, CD8 ⁺ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8 ⁺ cells. In some embodiments, the expression of phenotypic markers of central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO ⁺ , CD62L ⁺ , CD8 ⁺ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4 ⁺ T cells are further sorted into subpopulations. For example, CD4 ⁺ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

The immune cells, such as T cells, can be genetically modified following isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and/or expanded in vitro.

For example, cells can be transfected with the nucleic acid of the invention ex vivo. Various methods produce stable transfectants which express CARs of the invention. In one embodiment, a method of stably transfecting and re-directing cells is by electroporation using naked DNA. Additional methods to genetically engineer cells using naked DNA encoding a CAR of the invention include but are not limited to chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). The transfected cells demonstrating presence of an integrated un-rearranged vector and expression of the CAR may be expanded ex vivo. Viral transduction methods may also be used to generate redirected cells which express the CAR of the invention.

In another aspect, the invention relates to a method, e.g. an ex vivo or in vitro for producing a genetically modified cell or cell population comprising expressing in said cell or cell population a CAR nucleic acid construct of the invention. The method may include introducing into the cell a nucleic acid as described herein (e.g., an in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein). In embodiments, the RNA expresses the CAR polypeptide transiently. In one embodiment, the cell is a cell as described herein, e.g., an immune effector cell (e.g., T cells or NK cells, or cell population). Cells produced by such methods are also within the scope of the invention.

The present invention also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by poly A addition, to produce a construct containing 3' and 5' untranslated sequence (“UTR”), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR.

In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.

In some embodiments, the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines.

Cells of the invention may be cryopreserved such that the cells remain viable upon thawing. A fraction of the cells expressing the CARs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.

Cells described above can be used in adaptive immunotherapy for treatment of disease as further explained below. Pharmaceutical compositions

In another aspect of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid encoding a CAR as described herein, a vector comprising a nucleic acid encoding a CAR as described herein, a CAR as described herein or an isolated cell or cell population comprising a CAR according to the present invention and optionally a pharmaceutically acceptable carrier.

The genetically modified cells or pharmaceutical composition of the present invention can be administered by any convenient route, including parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Compositions can take the form of one or more dosage units.

The composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

The amount of the pharmaceutical composition of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

The compositions of the invention comprise an effective amount of a binding molecule of the present invention such that a suitable dosage will be obtained. The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

Typically, this amount is at least about 0.01 % of a binding molecule of the present invention by weight of the composition. Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present invention.

For intravenous administration, the composition can comprise from typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.

Suitable treatment with cells is also described below.

The present compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a binding molecule of the present invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

The pharmaceutical composition of the invention can be co-administered with other therapeutics, for example anti-cancer agents.

Methods of treatment MSLN is expressed on the surface of tumour cells and high expression levels of soluble MSLN have been correlated with poor prognosis in several cancers. Anti- MSLN antibodies have been investigated as anti-cancer therapeutics. These anti-MSLN antibodies either induce direct cell killing through their ADCC activity or are used in the form of ADCs.

The molecules and cells described herein are therefore expected to find application in the treatment of disease, in particular cancer.

In one embodiment, the disease is a disease associated with expression of mesothelin. The molecules of the invention may preferentially bind to MSLN present on the surface of a cancer cell as compared to soluble MSLN. The cancer to be treated using an antibody molecule of the invention therefore preferably expresses, or has been determined to express, MSLN. More preferably, cells of the cancer to be treated comprise, or have been determined to comprise,

MSLN at their cell surface, i.e. to comprise cell-surface bound MSLN. Methods for determining the presence of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

In one embodiment, the disease is cancer and the invention thus relates to methods for the prevention and/or treatment of cancer, comprising administering to a subject a cell or cell population comprising a CAR or a pharmaceutical formulation as described herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a cell and/or of a pharmaceutical composition of the invention.

The invention also relates to a CAR, a cell or cell population comprising a CAR a pharmaceutical formulation as described herein for use in therapy. The invention also relates to a CAR or a cell comprising a CAR a pharmaceutical formulation as described herein for use in the treatment of cancer. The invention also relates to the use of a CAR or a cell comprising a CAR a pharmaceutical formulation as described herein in the manufacture of a medicament for the treatment of cancer.

The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer includes all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of the histopathologic type or stage of invasiveness. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The cancer may be a primary or a secondary cancer. Thus, an antibody molecule as described herein may be for use in a method of treating cancer in an individual, wherein the cancer is a primary tumour and/or a tumour metastasis.

The cancer to be treated using an antibody molecule of the invention may be a solid cancer. Examples of various cancers are described further herein and include, but are not limited to, mesothelioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

The phrase "disease associated with expression of mesothelin" includes, but is not limited to, a disease associated with expression of mesothelin or condition associated with cells which express mesothelin including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a mesothelial hyperplasia; or a noncancer related indication associated with cells which express mesothelin. Examples of various cancers that express mesothelin include but are not limited to, mesothelioma, lung cancer, ovarian cancer, pancreatic cancer, and the like.

In another aspect, the invention relates to a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to a subject an effective amount of a cell or cell population that expresses a CAR of the invention, wherein the antigen binding domain is selected to specifically recognize the target cell population or tissue.

The term "stimulation," refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-p, and/or reorganization of cytoskeletal structures, and the like. In one embodiment, the cancer is selected from a haematological cancer or malignancy or a solid tumor. Hematologic cancers are cancers of the blood or bone marrow. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas.

In one embodiment, the cancer is metastatic.

Cancers that may be treated by methods, uses and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In one embodiment, the CAR binds to both MSLN and PSMA as described herein and is used to target PSMA and treat prostate cancer. Such a CAR includes an antigen binding domain specific to PSMA as described herein.

In therapies of prostatic disorders, e.g., prostate cancer, the therapy can be used in combination with existing therapies. In one embodiment, a CAR or cell comprising a CAR of the invention is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of a CAR-T or pharmaceutical composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the CAR-T or pharmaceutical composition of the invention is used in combination with surgery. The CAR-T or pharmaceutical composition of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.

In one embodiment, an immune checkpoint inhibitor is also administered with the cell or cell population or pharmaceutical composition. The immune checkpoint inhibitor may be an anti- PD1 , anti PDL-1 , anti PDL-2, anti CTL-4, anti-TIM-3 or anti LAG-3 antibody. In another embodiment, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, avelumab, durvalumab, or atezolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Pidilizumab, Toripalimab, Ipilimumab or Tremelimumab. In another embodiment, the immune checkpoint inhibitor is an interfering nucleic acid molecule, a small molecule or a PROteolysis TArgeting Chimera (PROTAC).

The immune checkpoint inhibitor may be administered before, after or at the same time as the cell pr cell population.

In another aspect, the invention relates to a method of providing an anti-tumor immunity in a subject, the method comprising administering to the mammal an effective amount of a cell or cell population genetically modified to express a CAR of the invention or a pharmaceutical formulation described herein, thereby providing an anti- tumor immunity in the subject.

All methods described above may be carried out in vivo, ex vivo or in vitro. Suitable treatment amounts of cells in the composition is generally at least 2 cells (for example, at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset) or is more typically greater than 10 ² cells, and up to 10 ⁶ , up to and including 10 ⁹ or 10 ⁹ cells and can be more than 1O ¹⁰ cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 10 ⁶ cells/ml and generally is greater than 10 ⁷ cells/ml, generally 10 ⁸ cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10®, 10 ¹ °, 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to the desired target antigen (MSLN), lower numbers of cells, in the range of I0 ⁶ /kilogram (10 ⁶ - 10 ¹¹ per patient) may be administered. CAR treatments may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy. The CAR expressing cell populations of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations

Kits and methods In another aspect, the invention provides a kit for detecting cancer, for example prostate cancer for diagnosis, treatment, prognosis or monitoring comprising a genetically modified cell or pharmaceutical composition of the invention. The kit may also comprise instructions for use. In one embodiment, the CAR-T or pharmaceutical composition comprises a label and one or more compounds for detecting the label. The invention in another aspect provides a binding molecule of the invention packaged in lyophilized form, or packaged in an aqueous medium.

In another aspect, the invention relates to a method of making a population of cells as described herein, the method comprising:

(i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells;

(ii) contacting the population of cells (for example, T cells) with the nucleic acid molecule of described herein, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and

(iii) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration. In one aspect, the disclosure provides a method of manufacturing an effective dose of engineered T cells for CAR T-cell therapy comprising: (a) preparing a population of engineered T cells comprising CAR described herein; (b) measuring the T cell expansion capability of the population; and (c) preparing an effective dose of engineered T cells for CAR T-cell therapy for treating a malignancy in a patient in need thereof based on the T cell expansion capability of the population. In some embodiments, the T cell expansion capability relates to in vivo expansion. In some embodiments, the T cell expansion capability relates to in vitro expansion. In some embodiments, the T cell expansion capability is measured during the manufacturing process. In another aspect, the disclosure provides a method of determining whether a patient will respond to the CAR T cell therapy comprising: (a) measuring in vivo CAR T-cell expansion after administration of CAR T-cells relative to pretreatment tumor burden to obtain a value and (b) determining if the patient will achieve durable response based on the value.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents and sequence database identifiers mentioned in this specification are incorporated herein by reference in their entirety.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The invention is further described in the non-limiting examples.

Examples

Materials and methods Construction of Tg/TKO mice

Mice carrying a heavy-chain antibody transgenic locus in germline configuration within a background that is silenced for endogenous heavy and light chain antibody expression (triple knock-out, or TKO) were created as previously described (WO2004/076618 and W02003/000737, Ren et al., Genomics, 84, 686, 2004; Zou et al., J. Immunol., 170, 1354, 2003, Teng et al ³⁰ all incorporated herein by reference). Briefly, transgenic mice were derived following pronuclear microinjection of freshly fertilised oocytes with a yeast artificial chromosome (YAC) comprising multiple human VH, D and J genes in combination with mouse immunoglobulin constant region genes lacking CH1 domains, mouse enhancer and regulatory regions. Yeast artificial chromosomes (YACs) are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells. The construction and use of YACs is well known in the art (e.g., Bruschi, C.V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002, Macmillan Publishers Ltd., Nature Publishing Group / www.els.net).

The YAC used comprised multiple human heavy chain V genes, human heavy chain D and J genes. It lacks the CH1 exon. The transgenic founder mice were back crossed with animals that lacked endogenous immunoglobulin expression to create the Tg/TKO lines used for immunisation with recombinant MSLN antigen.

Generation of VH domains

The Crescendo Mouse as described above was immunized with human PSMA and human MSLN recombinant proteins. Spleens and lymph nodes were harvested, cloned into a phagemid vector and selected by phage display. Libraries were generated from immunised mice and PSMA and MSLN binders were selected employing assays for target binding, including ELISA. Octet analysis was used to measure binding kinetics. For further description see WO 2017/122017, W02019/012260 and WO2017/191476. CAR construction

The following antigen-binding moieties were used: scFv derived from the J591 Ab specific for PSMA; human VH domain specific for PSMA (PSMA-VH); SCFV derived from a MSLN-specific Ab Amatuximab; human VH domain specific for MSLN (MSLN-VH). All ligands were assembled with the CD8a hinge and transmembrane domain, the CD28 costimulatory domain and CD3 intracellular signaling domain and cloned into the SFG retroviral vector. ²⁴ A FLAG-tag was incorporated after the antigen ligand to detect the expression of CARs by an anti-FLAG Ab. Dual specific (PSMA and MSLN) CARs were also generated by linking the two VH domains. The linkers used are described in more detail below. The corresponding CARs were called J591 , PSMA-VH, MSLN scFv, MSLN-VH and PSMA-VH/MSLN-VH. Retroviral supernatants were produced by transfection of 293 T cells with the retroviral vectors, the RD114 envelope from RDF plasmid and the MoMLV gag-pol from PegPam3-e plasmid. Supernatants were collected 48 hours and 72 hours after the transfection and filtered with 0.45 pm filter. ²⁴

The polypeptide sequences of the VH domains used were as follows:

MSLN-VH SEQ ID NO. 3 PSMA-VH SEQ ID NO. 18

PSMA-VH- (G ₄ S) ₃ -MSLN-VH SEQ ID NO. 505

EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSN KY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ GTMVTVSSGGGGSGGGGSGGGGSEITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGLGVG WIRQPPGKALEWLALIYWNDDKRYRPSLKNRLTIAKDTSKNQWLTMTNMDPVDTARYYCA HYSTSSETAFDI RGQGTMVTVSS

PSMA-VH- (G ₄ S) ₆ -MSLN-VH SEQ ID NO. 506

EVQLVESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKGLEWVAYISYDGSN KY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPAWGLRLGESSSYDFDIWGQ GTMVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEITLKESGPTLVKPTQTLTL

TCTFSGFSLSTSGLGVGWIRQPPGKALEWLALIYWNDDKRYRPSLKNRLTIAKDTSK NQWL TMTNM DPVDTARYYCAHYSTSSETAFDI RGQGTMVTVSS

Cell lines

Tumor cell lines PC-3, C4-2 (prostate cancer) and Aspc-1 (pancreatic cancer) were purchased from ATCC (American Type Culture Collection). All tumor cell lines were cultured with RPMI- 1640 (Gibco) supplemented with 10% Fetal bovine serum (Sigma), 2 mM GlutaMax (Gibco) and penicillin (100 units/mL) and streptomycin (100 g/mL; Gibco). All cells were cultured at 37°C with 5% CO2. PC-3 cell line was transduced with retroviral vectors encoding PSMA or MSLN to make PC-3-PSMA and PC-3-MSLN. PC-3-PSMA, PC-3-MSLN and Aspc-1 were transduced with retroviral vectors encoding Firefly-Luciferase-eGFP (FFIuc-eGFP) gene.

CAR-T cell generation

Buffy coats from healthy donors (Gulf Coast Regional Blood Center) were processed with Lymphoprep density separation (Fresenius Kabi Norge) to isolate peripheral blood mononuclear cells, which were then activated on plates coated with 1 pg/mL CD3 (Miltenyi Biotec) and 1 pg/mL CD28 (BD Biosciences) monoclonal Abs (mAbs). Two days later, activated T cells were transduced with retroviral supernatants on 24-well plates coated with retronectin (Takara Bio). T cells were collected 3 days after transduction and expanded in 40% RPMI-1640(Gibco) and 40% Click’s medium (Irvine Scientific), 10% HyClone FBS (GE healthcare), 2 mM GlutaMAX(Gibco), 100 unit/mL of Penicillin and 100 mg/mL of streptomycin (Gibco) with 10 ng/mL IL-7 (PeproTech) and 5 ng/mL IL-15 (PeproTech). T cells were collected for functional assays 12-14 days after activation. ^{25 26}

Flow cytometry mAbs for human CD3 (APC-H7; SK7; 560176), CD4 (BV711 ; SK3; 563028), CD8 (APC; SK1 ; 340584), CD45RA (PE; HI100; 555489), CD45RO (BV786; UCHL1 ; 564290), CD69 (FITC; L78; 347823), CCR7 (FITC; 150503; FAB197F-100), PD-1 (PE-Cy7; EH12.1 ;561272), Lag3(PE;T47-530;565616), FLAG (APC; L5; 637308), Granzyme-B (PE;GB11 ;561142) from BD biosciences and BioLegend were used. Samples were acquired with BD FACSCanto II or BD LSRFortessa. A minimum of 10000 events were acquired for each sample and were analyzed using FlowJo 10 (FlowJo).

Western blot

CAR-T cells were incubated with 2 pg anti-FLAG Ab in 100 pL PBS for 20 mins on ice and then with 2 pg goat antimouse secondary Ab for another 20 mins on ice. Cells were then incubated in the 37°C water bath for the selected time points and then lysed with 2 x Laemelli buffer for 10 mins. Cell lysates were then separated in 4% to 15% 10 well SDS-PAGE gels and transferred to polyvinylidene difluoride membranes at 75V for 120 mins (Bio-Rad). Blots were examined for human CD3 (Santa Cruz Biotechnology), p-Y142 CD3 (Abeam), pan- ERK (BD Biosciences), and pan-Akt, p-S473 Akt, and p-T202/Y204 MAPK (Cell Signaling Technology) with 1 :1000 dilution in 5% TBS-Tween milk. Membranes were incubated with HRP-conjugated secondary goat anti-mouse or goat anti-rabbit IgG (Santa Cruz) at a dilution of 1 :3000 and imaged with the ECL substrate kit (Thermofisher) on the ChemiDoc MP System (Bio-Rad) according to the manufacturer’s instructions. ²⁶

Proliferation assay T cells were labeled with 1.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) and plated with tumor cells at an effector to target (E:T) ratio of 1 :1. CFSE signal dilution from gated T cells on day 5 was measured using flow cytometry. ²⁶

In vitro cytotoxicity assay Tumor cells were seeded in 24-well plates at a concentration of 2.5x10 ⁵ cells/well overnight. CAR-T cells were added to the plate at an E:T of 1 :5 without exogenous cytokines. Cocultures were analyzed 5-7 days following coculture to measure residual tumor cells and T cells by flow cytometry. Dead cells were recognized by Zombie Aqua Dye (Biolegend) staining while CAR-T cells were identified by CD3 staining and tumor cells by GFP. ²⁶ CD69, PD-1 and Lag3 expression was measured by flow cytometry from day 0 to day 5 each day after coculture of CAR-T cells with tumor cells. For the granzyme-B staining, Golgi protein inhibitor (BD Biosciences) was added on day 1 of coculture for 6 hours. Cocultures were then first stained with Zombie Aqua Dye (Biolegend) and CD3 mAb, followed by fixation/permeabilization solution (BD Biosciences). Intracellular staining of granzyme-B was then conducted.

Cytokine analysis

CAR-T cells (1 xio ⁵ cells) were cocultured with 2.5x10 ⁵ tumor cells in 24-well plates without exogenous cytokines. Supernatant was collected after 24 hours, and cytokines (interferon-y (IFN-y) and IL-2) were measured by using ELISA kits (R&D, Research And Development system) in duplicates following manufacturer’s instructions. ²⁶

Expression and purification of recombinant proteins

A panel of recombinant proteins was produced, comprizing bispecific (2VH) proteins that bind both PSMA and MSLN, monospecific VH protein binding PSMA, monospecific VH protein binding MSLN and a control scFv protein based on Amatuximab. Bispecific protein was made in two formats, one with a short flexible linker (G4S)s, another one with a long flexible linker (G4S)e. Bispecific proteins were expressed in mammalian cells and purified by protein A binding. Monospecific proteins were His tagged at the C terminus, expressed in Escherichia coli and purified by His trap and size exclusion chromatography.

Binding and kinetic analysis

Binding analyses were performed at 25°C using BIAcore 8K system. The instrument was run on 1 x HBS-EP ⁺ (BR100669) buffer and the data were analyzed using Biacore Insight Evaluation software. Recombinant human MSLN was diluted to 2 ug/mL in 10 mM sodium acetate buffer pH4.0 and immobilized on a CM5 sensor chip (contact time 120 s) using amine- coupling kit with accordance to the manufacturer’s instructions. Humabody VH samples were tested for binding at 5 concentrations 3.7 nM, 11.1 nM, 33.3 nM, 100 nM and 300 nM using multicycle kinetics method. Each sample was injected for 100 s at the flow rate 35 pL/min and dissociated for 100 s. The antigen surface was regenerated by 20 s injection of 10 mM glycine pH 2.0. Recombinant human PSMA antigen with a human Fc tag was captured on a Protein G sensor. Humabody VH samples were tested in Single-cycle kinetics mode at increasing concentrations of 2.22 nM, 6.67 nM, 20 nM and 60 nM with 90 s association and 600 s dissociation time at the flow rate of 30 pL/min. Buffer injections were made to allow for doublereference subtraction. The sensor surface was regenerated with 10 mM glycine pH1.5 (GE Healthcare BR100354). To detect dual binding to MSLN and PSMA, human PSMA antigen surface was captured as above. Bispecific PSMA-MSLN Humabody constructs were captured on the PSMA surface by injecting 100 nM of each sample for 100 s at 35 pL/min flow rate. The capture was immediately followed by an injection of 300 nM recombinant human MSLN with 100 s contact time and 100 s dissociation. A PSMA-specific Humabody construct without a MSLN-binding arm was used as a control.

Xenograft murine models NSG (NOD scid gamma mouse) mice (6-8 weeks old) were injected intravenously through tail vein with either PC-3-PSMA-FFIuc-eGFP, or PC-3-PSMA-FFIuc-eGFP and PC-3-MSLN- FFIuc-eGFP mixed at 1 to 1 ratio, or Aspc-1-FFIuc-eGFP tumor cells of 1 xio ⁶ cells per mice. Fourteen days later, CAR-T cells were injected intravenously through tail vein. For the high dose treatment, 4x10 ⁶ CAR-T cells per mice were injected, while for the low dose treatment, 1 xio ⁶ CAR-T cells per mice were injected. In the rechallenge experiments, mice were infused 1 x 1 o ⁶ tumor cells per mice on clearance of the previous tumor. T umor growth was monitored by bioluminescence using IVIS (In Vivo Imaging Systems)-Kinetics Optical in vivo imaging system (PerkinElmer) (PSMA-VH and MSLN-VH part) or AMI (AMI Medical Imaging) Optical in vivo imaging system (Spectral instruments imaging) (PSMA-VH/MSLN-VH part). Statistics

All data was calculated and represented as mean with SD. One-way analysis of variance (ANOVA) or two-way ANOVA analyses were performed to compare multiple groups. Two- tailed t-test was used to compare two groups. P value of less than 0.05 was significant. All calculations and figures were achieved by GraphPad Prism V.7 (La Jolla, California, USA).

Results

Human VH domain-based CAR targeting PSMA is expressed and signals in T cells

We constructed the PSMA-specific CARs using the scFv from the J591 mAb (J591) and the PSMA binding human VH domain (PSMA-VH) joined to the CD8a stalk, CD28 costimulatory domain and CD3 intracellular domain. A flag-based tag was incorporated into the cassettes to detect CAR expression by flow cytometry (figure 1A). Activated T cells were successfully transduced and expressed the CARs equally (figure 1 B,C). The CD19-specific CAR (CD19) and non-transduced (NT) T cells were used as controls. On transduction, J591-T cells and PSMA-VH-T cells showed similar expansion in vitro when exposed to IL-15 and IL-7 cytokines, which was similar to CD19-T cells and NT-T cells (figure 1 D). Furthermore, no differences were observed in T cell composition as assessed by flow cytometry at day 12-14 of culture (figure 1 E). We examined proximal signaling of CAR-T cells before and after CAR cross-linking mediated by an anti-Flag Ab. Phosphorylation of the CAR-associated CD3 as well as phosphorylation of Akt and ERK were equal in J591-T cells and PSMA-VH-T cells (figure 1 F). Therefore, a VH domain-based CAR is expressed and signals in T cells on cross-linking as observed for scFv-based CAR-T cells.

PSMA-specific VH domain-based CAR-T cells are functional in vitro and in vivo

To compare the antitumor effect of PSMA-VH-T cells and J591-T cells in vitro, we engineered the PC3 cells to express PSMA antigen (figure 2A), and we observed that J591-T cells and PSMA-VH-T cells showed comparable granzyme-B expression when cultured with or without tumor cells (figure 2B, C and figure 8A). Similarly, both PSMA-VH-T cells and J591-T cells showed equal upregulation and subsequent down regulation of CD69 as a marker of T cell activation (figure 2D). Similar upregulation on antigen stimulation and down regulation after antigen removal were observed for PD-1 and Lag-3 ( figure 8B-E). We then cocultured PSMA- VH-T cells and J591-T cells in vitro with tumor cells (either PSMA-positive or PSMA-negative), and measured the remaining tumor cells after 5 days of coculture. CD19-T cells did not eliminate tumor cells, while PSMA-VH-T cells specifically eliminated PSMA-positive target cells (C4-2 and PC3-PSMA) to the same extent as conventional J591-T cells, and did not demonstrate off-target effect on PSMA-negative cells (PC3) (figure 2F,G). We also measured the secretion of IFNy and IL-2 after 24 hours of coculture with tumor cells. When the PSMA- VH-T and J591-T cells target PSMA-positive C4-2 and PC3-PSMA cells, both of them secreted high amount of IFNy and IL-2 compared with control CD19-CAR-T cells (figure 2H,I). Furthermore, PSMA-VH-T cells and J591-T cells proliferated similarly on encounter with tumor cells as shown by CFSE dilution (figure 2J). To investigate the antitumor effects of Humabody VH CAR-T cells in vivo, NSG mice engrafted with PSMA-positive tumor cells labeled with Firefly luciferase were treated with a high doses (4x10 ⁶ cells/mouse) of CAR-T cells (figure 3A). CAR- T cell treatment showed tumor control as measured by tumor bioluminescence without differences in mice treated with PSMA-VH-T cells or J591-T cells (figure 3B,C). To further assess differences between PSMA-VH-T cells and J591-T cells, we used low doses of T cells (1 xio ⁶ cells/mouse) in tumor-bearing mice (figure 3D). We observed that PSMA-VH-T cells still eliminated tumor cells in vivo as J591-T cells (figure 3E,F). In addition, we also observed similar VH CAR-T cell persistence in the peripheral blood, spleen and bone marrow compared with traditional scFv-based CAR-T cells at day 58 at the time of euthanasia (figure 3G,H). Therefore, Humabody VH CAR-T cells demonstrated comparable antitumor effects to scFv- based CAR-T cells in vitro and in vivo.

MSLN-specific VH domain-based CAR-T cells demonstrate antitumor activity To further assess the reproducibility of VH domain-based CARs, we tested a MSLN-specific Humabody VH. We constructed the conventional MSLN scFv CAR (MSLN-scFv) and VH domain CAR (MSLN-VH) using the same backbone developed for PSMA-specific CARs (figure 4A). MSLN-scFv and MSLN-VH were equally expressed in T cells ( figure 9A, B). In a similar fashion, we examined the antitumor activity of MSLN-scFv-T cells and MSLN-VH-T cells in vitro through coculture experiments with tumor cells, cytokine release assay, proliferation assay. MSLN-scFv-T cells and MSLN-VH-T cells selectively eliminated Aspc-1 tumor cells that express MSLN, while spared PC3 cells that do not express MSLN (figure 4B and figure 10). They released similar amount of IFNy and IL-2 (figure 4C) and proliferated on encounter with tumor cells (figure 4D). In the xenotransplant model in NSG mice engrafted with Aspc-1 cells labeled with Firefly luciferase (figure 4E), MSLN-VH-T cells showed even more profound antitumor effects as compared with mice treated with MSLN-scFv CAR-T cells (figure 4F,G), which translated in prolonged survival of the mice (figure 4H). However, we observed similar T cells expansion/persistence between MSLN-VH and MSLN-ScFv (figure 4I). Thus VH domain-based CARs can reproducibility redirect antitumor activity of engineered T cells.

In vitro analysis of monovalent and bivalent VH domain recombinant proteins

To test whether the VH domains are suitable to construct bispecific CARs, two VH domains in tandem recombinant proteins linking PSMA-specific and MSLN-specific VH were generated (figure 5A). To test whether the linkers had any effect on the target binding affinity, two different linkers were used: the (G4S)s linker (‘short flexible linker’) and a longer linker (G4S)e with 6 copies of the (G4S) repeat (‘long flexible linker’). Monomer VH proteins and a MSLN binding scFv were made as controls (figure 5A). Analysis of binding to PSMA recombinant protein by surface plasmon resonance (SPR) Biacore assay showed that the affinity of the PSMA-VH remained the same when the PSMA-VH was formatted with the MSLN-VH domain using either flexible linkers (figure 5B). Similarly, analysis of binding to MSLN recombinant protein by SPR Biacore assay showed that the affinity of the MSLN-VH domain was not altered when the PSMA-VH was formatted with the MSLN-VH using either flexible linkers (figure 5C). In summary, these data show that VH modules in bispecific format are capable of binding their specific target with the same affinity as their monovalent counterparts. Bispecific VH domain-based CAR-T cells demonstrate dual specificity

We constructed a bispecific VH domain CAR to facilitate CAR-T cells to specifically recognize two antigens simultaneously. We used the MSLN-VH and PSMA-VH domains fused with the short (G4S)s linker to generate the bispecific PSMA-VH/MSLN-VH CAR (figure 6A). The PSMA- VH/MSLN-VH CAR was expressed in T cells (figure 6B,C). PSMA-VH-T cells, MSLN-VH-T cells and PSMA-VH/MSLN-VH-T cells were cocultured with tumor the cell line Aspc-1 , which express MSLN, and the PC3-PSMA cell line. We observed the PSMA-VH/MSLN-VH-T can eliminate both tumor cell lines compared with single CAR-T cells, which only eliminate tumor cells expressing the targeted antigen (figure 11A, B). In addition, we also observed the expected cytokine release profile (figure 11C, D). Next, we confirmed that PSMA-VH/MSLN-VH-T cells displayed specific cytotoxicity toward the same cell line PC3 expressing either MSLN or PSMA similar to MSLN-VH-T cells and PSMA-VH-T cells without off-target effect (figure 6D,E). Importantly, when PC3-PSMA and PC3-MSLN were plated as 1 :1 ratio mixture in coculture experiments, only PSMA-VH/MSLN-VH-T cells fully eliminated the tumor cells, although PSMA- VH-T cells and MSLN-VH-T cells showed some bystander killing effect as previously observed ²⁷ (figure 6D,E). The in vitro antitumor effect was corroborated by release of IFN-y and IL-2 (figure 6F,G). To evaluate if bispecific VH domain CAR-T cells can eradicate tumors with mixed antigen expression in vivo, we established a metastatic xenograft mouse model by infusing PC3-PSMA cells and PC3-MSLN cells at 1 :1 ratio into NSG mice by intravenous injection. Mice were then treated with CAR19-T, PSMA-VH-T, MSLN-VH-T and PSMA-VH/MSLN-VH-T cells (figure 7A). Dual targeting PSMA-VH/MSLN-VH-T cells controlled the tumor growth more effectively than either single targeting PSMA-VH-T or MSLN-VH-T cells (figure 7B,C). CAR-T cells were detectable in the peripheral blood of these mice up to 4 weeks after infusion (figure 7D). We also observed that T cells expressing bispecific CAR showed similar phenotypic profile as single CAR targeting T cells for exhaustion and memory markers (figure 12A-C). Analyses of antigen expression in tumor cells in vivo showed that tumor cells growing in mice receiving either PSMA-VH-T or MSLN-VH-T cells were predominantly MSLN and PSMA expressing cells, respectively (figure 7E). These results indicate that bispecific CARs generated by joining two human Ab VH domains can prevent tumor escape in tumor with heterogeneous antigen expression.

Discussion

CARs approved by the Food and Drug Administration and those in clinical studies are mostly based on scFv-binding moieties. Here we demonstrated that monospecific human VH domainbased CAR-T cells achieved comparable antitumor effects both in vitro and in vivo as scFv- based CAR-T cells. Furthermore, VH domains combined in tandem to create bispecific molecules allowed the generation of effective CAR-T cells targeting two antigens. Redirected T cell based on single-domain Abs have been recently proposed. ^{17 28 29} However, most of them are obtained from llamas or camelid-derived libraries. Biological therapeutic molecules with non-human sequence can cause immune responses. ^{18 28} Transgenic mouse technology has enabled the generation of biophysically robust fully human VH domains known as Humabody VH or Humabodies ³⁰ which have the potential for use in CAR constructs while mitigating immunogenicity risk. Despite the remarkable clinical activity of CAR-T cells in hematological malignancies, objective responses in patients with solid tumors are modest. ^{10 26 31-33} Heterogeneity of antigen expression is one of the main reasons causing tumor escape in solid tumors after targeted therapies. ^{10 1920} Furthermore, murine-based scFv may cause immune responses especially in solid tumor patients who are usually less immunosuppressed compared with patients with liquid tumors. Targeting multiple TAAs and using human binding moiety in CAR molecules may improve the outcome of CAR-T cells in solid tumors. ¹⁰ Here, we demonstrated that human VH domains generated from a transgenic mouse might solve both issues of immunogenicity and tumor heterogeneity since bispecific CAR-T cells can be efficiently generated using two human VH domains in tandem.

In addition to the issue of heterogeneity in antigen expression, the complex inhibitory pathways of the tumor microenvironment in solid tumors mean that additional genetic modification of T cells would likely be required to enhance T cell trafficking and functions. ^{531 34-36} Generation of vector cassettes encoding multiple genes requires a significant optimization of the engineering strategies since the size of the entire cassette is limited. VH domains are a good alternative to scFv since they are approximately half the size.

Here, we have used two target antigens, PSMA and MSLN, that are currently under evaluation to treat mesothelioma, lung cancer, breast cancer, pancreatic cancer and prostate cancer via scFv-based CAR-T cells. ^37-39 Our preclinical experiments validate the potential use of bispecific human VH domains targeting both PSMA and MSLN in these difficult to treat malignancies. It remains to be validated if dual or multiple targeting with VH domain-based CARs can be broadly applicable, and if targeting multiple antigens in solid tumors leads to increased potential for toxicity.

Additionally, we observed that VH domain-based CAR-T cells have comparable cytotoxicity and proliferative capacity as traditional scFv-based CAR-T cells. MSLN-VH-T cells showed even more profound antitumor effects as compared with mice treated with MSLN-scFv CAR- T cells. Interestingly, MSLN-VH showed lower affinity than MSLN-scFv (28 nM compared with 79pM) recapitulating what has been observed for other scFvs that very high affinity is not necessarily optimal for CAR-based targeting for some targets. ^40-42 However, we cannot exclude that the observed superior antitumor activity of the MSLN-Vn-based CAR-T cells can be associated with the recognition of a different epitope rather than to different affinity. In summary, we have demonstrated that VH domain CAR-T cells in monospecific format achieved comparable antitumor response compared with traditional scFv-based CAR-T cells both in vitro and in vivo. Furthermore, bispecific VH domain CAR-T cells delivered potent anti-tumor effects demonstrating the potential to target solid tumors with heterogeneous antigen expression. These proof-of-concept experiments lay the foundation for further development of human VH domain-based CAR-T cells in clinical trials.

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