阅读说明：本技术 表达嵌合活化受体及嵌合刺激受体的细胞及其用途 (Cells expressing chimeric activating receptors and chimeric stimulating receptors and uses thereof ) 是由 刘宏 张鹏博 L·霍兰 许奕阳 B·K·斯特利 刘连兴 云泓若 于 2018-04-24 设计创作，主要内容包括：本申请案提供免疫细胞(诸如T细胞),其包括嵌合抗体-T细胞受体TCR构筑体caTCR及嵌合信号传导受体CSR构筑体。所述caTCR包括特异性结合于靶抗原的抗原结合模块及能够募集至少一种TCR相关信号传导分子的T细胞受体模块TCRM,且所述CSR包括特异性结合于靶配体的配体结合域及能够提供刺激信号至所述免疫细胞的协同刺激信号传导域。还提供制造及使用这些细胞的方法。(The present application provides immune cells (such as T cells) comprising a chimeric antibody-T cell receptor TCR construct, caTCR, and a chimeric signaling receptor CSR construct. The calcr comprises an antigen binding moiety that specifically binds to a target antigen and a T cell receptor moiety TCRM capable of recruiting at least one TCR-associated signaling molecule, and the CSR comprises a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain capable of providing a stimulatory signal to the immune cell. Methods of making and using these cells are also provided.)

1. An immune cell, comprising:

a) a chimeric antibody-T cell receptor TCR construct, caTCR, comprising:

i) an antigen binding moiety that specifically binds to a target antigen; and

ii) a T cell receptor module TCRM comprising a first TCR domain TCRD comprising a first TCR transmembrane domain TCR-TM and a second TCRD comprising a second TCR-TM, wherein the TCRM contributes to recruitment of at least one TCR-related signaling molecule; and

b) a chimeric signaling receptor CSR comprising:

i) a ligand binding module capable of binding to or interacting with a target ligand;

ii) a transmembrane module; and

iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to the immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain.

2. The immune cell of claim 1, wherein the CSR lacks any primary immune cell signaling sequence.

3. The immune cell of claim 1 or2, wherein the target antigen is a cell surface antigen.

4. The immune cell of claim 1 or2, wherein the target antigen is a complex comprising a peptide and a Major Histocompatibility Complex (MHC) protein.

5. The immune cell of any one of claims 1-4, wherein the first TCR-TM is derived from one of the transmembrane domains of a first T cell receptor and the second TCR-TM is derived from the other transmembrane domain of the first T cell receptor.

6. The immune cell of claim 5, wherein at least one of the TCR-TMs is non-naturally occurring.

7. The immune cell of claim 5 or 6, wherein the first T cell receptor is a gamma/delta T cell receptor.

8. The immune cell of any one of claims 1-7, wherein the antigen binding moiety is multispecific.

9. The immune cell of any one of claims 1-8, wherein the caTCR further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the caTCR.

10. The immune cell of claim 9, wherein the stabilizing moiety is selected from the group consisting of: c_H1-C_LModule, C_H2-C_H2 Module, C_H3-C_H3 Module and C_H4-C_HAnd 4, modules.

11. The immune cell of any one of claims 1-10, wherein the target antigen and the target ligand are the same.

12. The immune cell of any one of claims 1-10, wherein the target antigen and the target ligand are different.

13. The immune cell of claim 12, wherein the target ligand is a ligand expressed on the surface of a cell presenting the target antigen.

14. The immune cell of any one of claims 1-13, wherein the target ligand is a disease-associated ligand.

15. The immune cell of claim 14, wherein the target ligand is a cancer-associated ligand.

16. The immune cell of claim 14, wherein the target ligand is a virus-associated ligand.

17. The immune cell of any one of claims 1-13, wherein the target ligand is an immunomodulatory molecule.

18. The immune cell of claim 17, wherein the immunomodulatory molecule is an immunosuppressive receptor and the CSR is an antagonist of the immunosuppressive receptor.

19. The immune cell of claim 17, wherein the immunomodulatory molecule is an immunostimulatory receptor and the CSR is an agonist of the immunostimulatory receptor.

20. The immune cell of claim 17, wherein the target ligand is an immune checkpoint molecule.

21. The immune cell of claim 17, wherein the target ligand is an inhibitory cytokine.

22. The immune cell of any one of claims 1-13, wherein the target ligand is an apoptotic molecule.

23. The immune cell of any one of claims 1-22, wherein the ligand binding moiety is an antibody moiety.

24. The immune cell of any one of claims 1-22, wherein the ligand binding moiety is derived from an extracellular domain of a receptor.

25. The immune cell of any one of claims 1-24, wherein the transmembrane module of the CSR comprises a transmembrane domain derived from CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154.

26. The immune cell of any one of claims 1-25, wherein the co-stimulatory immune cell signaling module is derived from the intracellular domain of a co-stimulatory receptor of a TCR.

27. The immune cell of claim 26, wherein the co-stimulatory receptor is selected from the group consisting of: CD28, 4-1BB, OX40, ICOS, CD27 and CD 40.

28. The immune cell of any one of claims 1-27, wherein expression of the CSR is inducible.

29. The immune cell of claim 28, wherein the expression of the CSR is inducible upon activation of the immune cell.

30. The immune cell of any one of claims 1-29, wherein the antigen-binding moiety of the cadR comprises an antibody moiety that binds to CD19, wherein the ligand-binding moiety of the CSR comprises a scFv that binds to CD19, and wherein the transmembrane moiety and costimulatory immune cell signaling moiety are both derived from CD 28.

31. The immune cell of any one of claims 1-29, wherein the antigen-binding moiety of the caTCR comprises an antibody moiety that binds to AFP, wherein the ligand-binding moiety of the CSR comprises an scFv that binds to GPC3, and wherein the transmembrane moiety and costimulatory immune cell signaling moiety are both derived from CD 28.

32. The immune cell of any one of claims 1-29, wherein the antigen-binding moiety of the cadR comprises an antibody moiety that binds to CD19, wherein the ligand-binding moiety of the CSR comprises a scFv that binds to CD20, and wherein the transmembrane moiety and costimulatory immune cell signaling moiety are both derived from CD 28.

33. One or more nucleic acids encoding a caTCR and CSR according to any of claims 1-32, wherein the caTCR and CSR are each comprised of one or more polypeptide chains encoded by the one or more nucleic acids.

34. One or more vectors comprising one or more nucleic acids according to claim 33.

35. An immune cell comprising one or more nucleic acids according to claim 33, one or more vectors according to claim 34.

36. The immune cell of any one of claims 1-32, wherein the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

37. A pharmaceutical composition comprising the immune cell of any one of claims 1-32 and a pharmaceutically acceptable carrier.

38. A method of killing a target cell presenting a target antigen (or treating a target antigen-associated disease), comprising contacting the target cell with an immune cell of any one of claims 1-32.

39. A method of treating a target antigen-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 37.

40. A method of providing a costimulatory signal to an immune cell comprising a caTCR or transduced with a nucleic acid encoding a caTCR comprising introducing into the cell one or more nucleic acids according to claim 33 or one or more vectors according to claim 34.

Technical Field

The invention relates to immune cells, such as T cells, that include a chimeric antibody-T Cell Receptor (TCR) construct (caTCR) and a Chimeric Signaling Receptor (CSR) construct. The caTCR includes an antigen binding moiety that specifically binds to a target antigen and a T Cell Receptor Moiety (TCRM) capable of recruiting at least one TCR-associated signaling molecule, and the CSR includes a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain capable of providing a stimulatory signal to the immune cell.

Background

T cell mediated immunity is the adaptive process of developing antigen (Ag) -specific T lymphocytes to eliminate viral, bacterial, parasitic infections or malignant cells. It may also involve abnormal recognition of self-antigens, leading to autoimmune inflammatory diseases. Ag specificity of T lymphocytes is based on the recognition by the T Cell Receptor (TCR) of unique antigenic peptides presented by Major Histocompatibility Complex (MHC) molecules on Ag Presenting Cells (APC) (Broere et al, Principles of immunopharmacology, 2011). Due to developmental selection after maturation in the thymus, each T lymphocyte expresses a unique TCR on the cell surface. TCRs exist in two forms: an α β heterodimer or a γ δ heterodimer. T cells express TCRs in either the α β form or the γ δ form on the cell surface. The four chains α/β/γ/δ all have characteristic extracellular structures consisting of a highly polymorphic "immunoglobulin variable region" -like N-terminal domain and an "immunoglobulin constant region" -like second domain. Each of these domains has a characteristic intra-domain disulfide bridge. The constant region is adjacent to the cell membrane, followed by a connecting peptide, a transmembrane region, and a short cytoplasmic tail. The covalent bond between The 2 chains of a heterodimeric TCR is formed by a cysteine residue located within a short connecting peptide sequence bridging The extracellular constant domain and The transmembrane region, which cysteine residue forms a disulfide bond with The mating TCR chain cysteine residue at The corresponding position (The T cell Receptor facersbook, 2001).

The α β and γ δ TCRs bind to non-polymorphic membrane-bound CD3 protein to form a functional octameric TCR-CD3 complex, consisting of a TCR heterodimer and three dimeric signalling molecules CD3 δ/epsilon, CD3 γ/epsilon, and CD3 ζ/ζ or ζ/η. The ionizable residues in the transmembrane domain of each subunit form a polar interaction network that holds the complexes together. With respect to T cell activation, The variable region at The N-terminus of The TCR recognizes a peptide/MHC complex presented on The surface of The target cell, whereas The CD3 protein is involved in signal transduction (Call et al, cell.111(7):967-79, 2002; The T cell Receptor facebook, 2001).

α β TCRs, also known as conventional TCRs, are expressed on most lymphocytes and consist of glycosylated polymorphic α and β chains. Different α β TCRs can distinguish between different peptides embedded in the surface of MHC II (mostly expressed on the surface of APC cells) and MHC I (expressed on all nucleated cells) molecules, which are relatively constant in size and shape. Gamma Delta TCR, although structurally similar to α β TCR, recognizes carbohydrate, nucleotide or phosphor-bearing antigens in a manner unrelated to MHC presentation (The T cell receptor facebook, 2001; Girardi et al, J.invest.Dermatol.126(1):25-31,2006; Hayes et al, Immunity.16(6):827-38, 2002).

Over the past two decades, fundamental advances in immunology and tumor biology and the identification of a large number of tumor antigens have contributed to significant advances in the field of cell-based immunotherapy. In the field of cell-based immunotherapy, T cell therapy has taken an important position, with the goal of treating cancer by transferring autologous and ex vivo expanded T cells to patients, and some notable anti-tumor responses have been generated (Blattman et al, science.305(5681):200-5, 2004). For example, in melanoma patients, including large invasive tumors, administration of ex vivo expanded naturally occurring Tumor Infiltrating Lymphocytes (TILs) at multiple sites including liver, lung, soft tissue and brain can mediate objective response rates in the range of 50-70% (Rosenberg et al, nat. Rev. cancer.8(4): 299-.

A major limitation of the widespread use of TIL therapy is the difficulty in generating human T cells with anti-tumor potential. Alternatively, exogenous high affinity TCRs can be introduced into normal autologous T cells of a patient via T cell engineering. The permissive metastasis of these cells to lymphodeficient patients has been shown to mediate cancer regression in cancers such as melanoma, colorectal cancer, and synovial sarcoma (Kunert R et al, front. Immunol.4:363,2013). The latest phase I clinical trial used anti-NY-ESO-1 TCR against synovial sarcoma, reporting an overall response rate of 66% and achieving a complete response in one patient receiving T cell therapy (Robbins PF et al, Clin. cancer Res.21(5):1019-27, 2015).

One of the advantages of TCR-engineered T cell therapy is that it can target entire arrays of potentially intracellular tumor-specific proteins that are processed and delivered to the cell surface via MHC presentation. In addition, TCRs are highly sensitive and can be activated by only a few antigenic peptide/MHC molecules, which in turn can trigger cytolytic T cell responses, including cytokine secretion, T cell proliferation, and limiting cytolysis of target cells. Thus, TCR-engineered T cells are particularly valuable for their ability to kill target cells with minimal copies of intracellular target antigens, as compared to antibody or small molecule therapies (Kunert R et al, front.

However, unlike therapeutic antibodies, which are most likely discovered via hybridoma or presentation techniques, identification of target-specific TCRs requires establishment of target peptide/MHC-specific TCR clones from patient T cells and screening for the correct α - β chain combination with optimal target antigen binding affinity. Phage/yeast display is often employed after cloning of the TCR from the patient's T cells to further enhance the target binding affinity of the TCR. The entire process requires expertise in many areas and is time consuming (Kobayashi et al, Oncoimmunology.3(1): E27258,2014). Difficulties in TCR exploration have greatly hindered the widespread use of TCR-engineered T cell therapies. It is also hampered by treatment-related toxicity, especially TCRs directed against antigens that are overexpressed on tumor cells and also expressed on healthy cells, or TCRs that recognize off-target peptide/MHC complexes (Rosenberg SA et al, science.348(6230):62-8,2015).

A different approach has been developed in recent years to involve T cells in targeted cancer immunotherapy. This new approach is referred to as chimeric antigen receptor T cell therapy (CAR-T). Which combines the sharp targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. CARs are composed of an extracellular domain that recognizes a cell surface antigen, a transmembrane region, and an intracellular signaling domain. The extracellular domain consists of antigen-binding variable regions from the heavy and light chains of a monoclonal antibody fused into single chain variable fragments (scfvs). The intracellular signaling domain contains immunoreceptor tyrosine-based activation motifs (ITAMs), such as those from CD3 ζ or FcR γ, and one or more costimulatory signaling domains, such as those from CD28, 4-1BB or OX40 (Barrett DM et al, Annu. Rev. Med.65:333-47, 2014; Davila ML et al, Oncoimunology.1 (9):1577-1583, 2012). Binding of the CAR to the target antigen grafted onto the surface of the T cell can trigger T cell effector functions unrelated to TCR-peptide/MHC complex interactions. Thus, T cells with CARs can be redirected to attack a variety of cells, including those that do not match the MHC type of the TCR on the T cell but express the target cell surface antigen. This approach overcomes the constraints of MHC-restricted TCR recognition and avoids tumor escape via antigen presentation or impairment of MHC molecule expression. Clinical trials have shown that CAR-T therapies have clinically significant anti-tumor activity in neuroblastoma (Louis CU et al, blood.118(23):6050-6056,2011), B-ALL (Maude, SL et al, New England Journal of Medicine 371:16: 1507-. In one study, complete remission rates of 90% in 30 patients with B-ALL treated with CD19-CAR T therapy were reported (Maude, SL et al, supra).

All CAR studies to date have been directed to tumor antigens with high cell surface expression. To target low copy number cell surface tumor antigens and intracellular tumor antigens, which represent 95% of all known tumor-specific antigens, it is necessary to develop more potent and more effective engineered cell therapies (Cheever et al, clin. cancer res.15(17):5323-37, 2009).

Several attempts have been made to engineer chimeric receptor molecules with antibody specificity and T cell receptor effector functions. See, e.g., Kuwana, Y et al, biochem. Biophys. Res. Commun.149(3):960-968, 1987; gross, G et al, Proc.Natl.Acad.Sci.USA.86: 10024-; gross, G and Eshhar, Z, FASEB J.6(15):3370-3378, 1992; and U.S. patent No. 7,741,465. To date, none of these chimeric receptors are for clinical use, and there is a need for novel designs for antibody-TCR chimeric receptors with improved expression and function in human T cells.

The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. PCT application No. PCT/US2016/058305 is hereby incorporated by reference in its entirety.

Disclosure of Invention

The present application provides in one aspect immune cells, such as T cells, that include a chimeric antibody-T Cell Receptor (TCR) construct (caTCR) and a Chimeric Signaling Receptor (CSR) construct. The caTCR includes an antigen binding moiety that specifically binds to a target antigen and a T Cell Receptor Moiety (TCRM) capable of recruiting at least one TCR-associated signaling molecule, and the CSR includes a ligand binding domain that specifically binds to a target ligand and a costimulatory signaling domain capable of providing a stimulatory signal to the immune cell.

In some embodiments, there is provided an immune cell comprising: a) a chimeric antibody-T Cell Receptor (TCR) construct (caTCR) comprising i) an antigen binding moiety that specifically binds to a target antigen; and ii) a T Cell Receptor Module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM contributes to recruitment of at least one TCR-related signaling molecule; and b) a Chimeric Signaling Receptor (CSR) comprising: i) a ligand binding module capable of binding to and interacting with a target ligand; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to the immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain.

In some embodiments, one or more nucleic acids are provided that encode a caTCR and a CSR as described herein, wherein each of the caTCR and the CSR consists of one or more polypeptide chains encoded by the one or more nucleic acids.

In some embodiments, one or more nucleic acids are provided that encode: a) a chimeric antibody-T Cell Receptor (TCR) construct (caTCR), comprising: i) an antigen binding moiety that specifically binds to a target antigen; and ii) a T Cell Receptor Module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM contributes to recruiting of at least one TCR-related signaling molecule, and wherein the caTCR is comprised of one or more polypeptide chains; and b) a Chimeric Signaling Receptor (CSR) comprising: i) a ligand binding module capable of binding to or interacting with a target ligand; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to an immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, wherein the CSR lacks a functional primary immune cell signaling domain, and wherein the CSR consists of one or more polypeptide chains.

In some embodiments, one or more vectors are provided that include one or more nucleic acids as described herein.

In some embodiments, a composition is provided that includes one or more nucleic acids or one or more vectors as described herein.

In some embodiments, an immune cell is provided that includes one or more nucleic acids or one or more vectors as described herein.

In some embodiments, a pharmaceutical composition is provided that includes an immune cell as described herein and a pharmaceutically acceptable carrier.

In some embodiments, a method of killing a target cell presenting a target antigen is provided, comprising contacting the target cell with an immune cell described herein.

In some embodiments, there is provided a method of treating a target antigen associated disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.

In some embodiments, a method of providing a costimulatory signal to an immune cell comprising a caTCR or transduced with a nucleic acid encoding a caTCR is provided, comprising introducing into the cell one or more nucleic acids or one or more vectors as described herein.

Drawings

FIG. 1 shows a schematic representation of several caTCR molecules with different antigen binding modules.

FIG. 2 shows NALM6(CD 19) from a killed cancer cell line⁺) The killing is mediated by T cells transduced with anti-CD 19 caTCR-1-0 alone, anti-CD 19CSR alone, or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 19 CSR.

FIG. 3 shows the concentrations of cytokines (IL-2, GM-CSF, IFN- γ, and TNF- α) found in supernatants following in vitro killing of cancer cell line NALM6 mediated by T cells transduced with anti-CD 19 caTCR-1-0 alone, anti-CD 19CSR alone, or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 19 CSR.

FIG. 4 shows degranulation activity (as determined by CD107a expression) in T cells transduced with anti-CD 19 caTCR-1-0 alone, anti-CD 19CSR alone, or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 19CSR after stimulation with the cancer cell line NALM 6.

FIG. 5 shows the proliferation (as determined by CFSE dye dilution) of T cells transduced with anti-CD 19 caTCR-1-0 or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 19CSR alone following stimulation with cancer cell lines BV173 or NALM 6.

FIG. 6 shows killing cancer cell lines HepG2 (AFP)⁺/GPC3⁺) And HepG2-GPC3.ko (AFP)⁺/GPC3^-) The killing is mediated by T cells transduced with anti-AFP 158/HLA-A2: 01caTCR-1-0 alone, anti-GPC 3CSR alone or anti-AFP 158/HLA-A2: 01caTCR (1-0 and 1-TM5) + anti-GPC 3 CSR.

FIG. 7 shows the concentrations of cytokines (IL-2, GM-CSF, IFN-. gamma.and TNF-. alpha.) found in the supernatants following in vitro killing of cancer cell lines HepG2 and HepG2-GPC3.ko mediated by T cells transduced with anti-AFP 158/HLA-A2: 01caTCR-1-0, anti-GPC 3CSR alone or anti-AFP 158/HLA-A2: 01caTCR (1-0 and 1-TM5) + anti-GPC 3 CSR.

FIG. 8 shows degranulation activity (as determined by CD107a expression) in T cells transduced with anti-AFP 158/HLA-A2: 01caTCR-1-0 alone, anti-GPC 3CSR alone, or anti-AFP 158/HLA-A2: 01caTCR (1-0 and 1-TM5) + anti-GPC 3CSR alone following stimulation with cancer cell line HepG 2.

FIG. 9 shows the proliferation (as determined by CFSE dye dilution) of T cells transduced with anti-AFP 158/HLA-A2: 01caTCR-1-0 alone, anti-GPC 3CSR alone, or anti-AFP 158/HLA-A2: 01caTCR (1-0 and 1-TM5) + anti-GPC 3CSR after stimulation with cancer cell line HepG 2.

FIG. 10 shows the killing of cancer cell lines Raji, BV173, NALM6 and Jeko-1 (each CD 19)⁺/CD20⁺) The killing is mediated by T cells transduced with anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 20 CSR.

FIG. 11 shows the concentrations of cytokines (IL-2, GM-CSF, IFN-. gamma.and TNF-. alpha.) found in the supernatants following in vitro killing of cancer cell lines Raji mediated by T cells transduced with anti-CD 19 caTCR-1-0 or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 20CSR alone.

FIG. 12 shows the degranulation activity of T cells transduced with anti-CD 19 caTCR-1-0 alone, anti-CD 20CSR alone, or anti-CD 19 caTCR (1-0 and 1-TM5) + anti-CD 20CSR (as determined by CD107a expression) following stimulation with cancer cell line Raji.

FIGS. 13A-13E show schematic structures of exemplary bispecific caTCR molecules.

FIGS. 14 to 15 show the proliferation of T cells expressing anti-CD 19 caTCR-1-0 and anti-CD 19CSR (as determined by CFSE dye dilution) following stimulation with the cancer cell line NALM 6.

Figure 16 shows tumor growth of NALM6 in a subcutaneous mouse model, mock-treated or single intratumoral injection of T cells expressing anti-CD 19 caTCR-1 or anti-CD 19 caTCR-1 in combination with anti-CD 19 CSR-1.

Figure 17 shows serum cytokine levels in mice treated with T cells expressing anti-CD 19 CAR or T cells expressing both anti-CD 19 caTCR-1 and anti-CD 19 CSR-1. The sautington t-test was used to analyze the statistical significance of serum cytokine levels in both groups (. P < 0.01;. P < 0.001;. P < 0.0001).

Figure 18 shows tumor growth of HepG2 in a subcutaneous mouse model, which was mock-treated or single intratumoral injection of T cells expressing anti-AFP CAR or anti-AFP CAR in combination with anti-GPC 3 CSR.

Detailed Description

The present application provides immune cells, such as T cells, that include a chimeric antibody-T Cell Receptor (TCR) construct (caTCR) and a Chimeric Signaling Receptor (CSR) construct. The caTCR includes an antigen binding module that specifically binds to a target antigen and a T Cell Receptor Module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. The CSR comprises a ligand binding domain that specifically binds to a target ligand and a co-stimulatory immune cell signaling domain capable of providing a stimulatory signal to an immune cell, and does not comprise a functional primary immune cell signaling sequence. The target antigen and target ligand may be proteins expressed on the surface of cells or complexes comprising peptides and MHC proteins (referred to herein as "pMHC complexes" or "pMHC"), such as MHC-presented disease-associated antigenic peptides on the surface of diseased cells. The caTCR is regulated by a naturally occurring mechanism that controls T cell receptor activation, and signaling via the CSR can enhance the immune response mediated by the caTCR.

A series of novel T cells have been developed that include the caTCR and CSR constructs. It exhibits strong cytotoxicity against tumor cells bearing the target, wherein cytokine expression in response to target cell engagement is increased compared to cells expressing only the caTCR without the presence of CSR. The inclusion of CSR in these cells further enhanced degranulation, proliferation and viability compared to cells expressing caTCR only.

The present application thus provides an immune cell comprising a calcr specific for a target antigen and a CSR specific for a target ligand, wherein the calcr comprises an antigen binding module that specifically binds to a target antigen and a T Cell Receptor Module (TCRM) capable of recruiting at least one TCR-associated signaling molecule, and wherein the CSR: a) involving ligand binding specifically to a target ligandA domain and a co-stimulatory immune cell signaling domain capable of providing a stimulatory signal to an immune cell, and b) does not include a functional primary immune cell signaling sequence. The caTCR can employ any of a variety of patterns of antigen binding modules and/or TCRM variations. For example, the caTCR may have: an antigen binding module comprising a peptide selected from the group consisting of Fab, Fab ', (Fab')₂Fv, and scFv; and a TCRM comprising one or more sequences derived from an α/β or γ/δ TCR, the one or more sequences comprising a variant in one or more of a transmembrane domain, a connecting peptide, and an intracellular domain. See fig. 1. In some embodiments, the antigen binding moiety is multispecific (such as bispecific). The CSR can similarly have a ligand binding moiety comprising a ligand selected from the group consisting of Fab, Fab ', (Fab')₂Fv, and scFv. In some embodiments, the target antigen is the same as the target ligand. In some embodiments, the antigen binding moiety of the caTCR is the same as the ligand binding moiety of the CSR, or comprises the same sequences, such as CDRs or variable domains, that confer antigen specificity. In some embodiments, the target antigen is different from the target ligand.

In other aspects, there is provided: a) one or more nucleic acids encoding a caTCR and CSR; b) an immune cell comprising one or more nucleic acids encoding a caTCR and a CSR, and c) a composition comprising an immune cell comprising i) a caTCR and a CSR or ii) one or more nucleic acids encoding a caTCR and a CSR. The composition may be a pharmaceutical composition.

Also provided are methods of making immune cells comprising caTCR and CSR and using them for therapeutic uses, as well as kits and articles of manufacture suitable for use in these methods. Further provided are methods of treating diseases using immune cells comprising a caTCR and CSR.

Definition of

The term "antibody" or "antibody portion" encompasses full-length antibodies and antigen-binding fragments thereof. Full-length antibodies comprise two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops called Complementarity Determining Regions (CDRs) (light chain (LC) CDRs comprise LC-CDR1, LC-CDR2 and LC-CDR3, and Heavy Chain (HC) CDRs comprise HC-CDR1, HC-CDR2 and HC-CDR 3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani convention (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking extensions called Framework Regions (FRs) that are more highly conserved than the CDRs and form an architecture that supports hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified into various classes based on the amino acid sequence of the constant region of their heavy chains. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG and IgM, characterized by the presence of alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes fall into subclasses, such as lgG1(γ 1 heavy chain), lgG2(γ 2 heavy chain), lgG3(γ 3 heavy chain), lgG4(γ 4 heavy chain), lgA1(α 1 heavy chain), or lgA2(α 2 heavy chain).

The term "antigen-binding fragment" as used herein refers to an antibody fragment comprising, for example, a diabody, a Fab ', a F (ab ')2, a Fv fragment, a disulfide stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv '), a disulfide stabilized diabody (ds diabody), a single chain antibody molecule (scFv), a scFv dimer (diabody), a multispecific antibody formed from a portion of an antibody that includes one or more CDRs, a camelid single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not include an intact antibody structure. The antigen binding fragment is capable of binding to the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv) binds. In some embodiments, an antigen-binding fragment can include one or more CDRs from a particular human antibody grafted to framework regions from one or more different human antibodies.

As used herein, a first antibody moiety "competes" for binding to a target antigen with a second antibody moiety when the first antibody moiety inhibits binding of the target antigen of the second antibody moiety by at least about 50% (such as at least about any one of: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the first antibody moiety, or vice versa. High throughput methods for "grouping" antibodies based on their cross-competition are described in PCT publication No. WO 03/48731.

As used herein, the term "specific binding" or "specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, which determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody moiety that specifically binds to a target (which may be an epitope) is one that binds to a target with higher affinity, avidity, more readily, and/or for longer duration than it binds to other targets. In some embodiments, the portion of the antibody that specifically binds to the antigen reacts with one or more antigenic determinants of the antigen (e.g., a cell surface antigen or a peptide/MHC protein complex) with a binding affinity of at least about 10-fold greater than its binding affinity for other targets.

The term "T cell receptor" or "TCR" refers to a heterodimeric receptor consisting of paired α β or γ δ chains on the surface of a T cell. Each α, β, γ and δ chain is composed of two Ig-like domains: a variable domain (V) conferring antigen recognition via a Complementarity Determining Region (CDR), followed by a constant domain (C) anchored to the cell membrane by a connecting peptide and a Transmembrane (TM) region. The TM region binds to a constant subunit of the CD3 signaling device. The V domains each have three CDRs. Complex interactions between these CDRs and antigenic peptides bound to proteins encoded by the major histocompatibility Complex (pMHC) (Davis and Bjorkman (1988) Nature,334, 395-402; Davis et al (1998) Annu Rev Immunol,16, 523-544; Murphy (2012), xix, p.868).

The term "TCR-associated signaling molecule" refers to a molecule having the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), which is part of the TCR-CD3 complex. TCR-related signaling molecules include CD3 γ epsilon, CD3 δ epsilon, and ζ ζ ζ (also referred to as CD3 ζ or CD3 ζ ζ).

"activation" as used herein with respect to cells expressing CD3 refers to a state of the cell that has been sufficiently stimulated to induce a detectable increase in downstream effector functions of the CD3 signaling pathway, including but not limited to cell proliferation and cytokine production.

The term "module" when referring to a portion of a protein is meant to encompass structurally and/or functionally related portions of one or more polypeptides that make up the protein. For example, the transmembrane module of a dimeric receptor may refer to the transmembrane portion of each polypeptide chain of the receptor. Modules may also refer to the relevant portions of a single polypeptide chain. For example, a transmembrane module of a monomeric receptor can refer to the transmembrane portion of a single polypeptide chain of the receptor. Modules may also comprise only a single portion of a polypeptide.

The term "isolated nucleic acid" as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin, or some combination thereof, by virtue of which the "isolated nucleic acid" (1) does not bind to all or a portion of a polynucleotide in nature to which it is found, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

As used herein, the terms "CDR" or "complementarity determining region" are intended to mean the non-contiguous antigen combining sites present within the variable regions of both heavy and light chain polypeptides, such specific regions are described in Kabat et Al, J.biol.chem.252: 6609-internest (1977); Kabat et Al, U.S. department of health and Human Services (U.S. Dept. of health and Human Services), "immunoglobulin Sequences of interest (Sequences of proteins of immunological interest)" (1991); Chothia et Al, J.mol.biol.196:901-917 (1987); Al-Lazikani B. et Al, J.mol.biol.,273: 948(1997) -pluum et Al, J.mol.262: 745 (262: Abhin) and/or in the context of the methods described herein, such references include the definitions of the amino Acids cited in the references cited herein, and the references in the various references cited herein, such as the Sequences of antibodies, such as cited in the Japanese-10. dockerin, published application, including the CDR Sequences of the aforementioned, published application, published by the methods, published application, and the references cited in the various references cited herein, such as the various references, including the Sequences of the various references cited in the fields of the methods of the application, such references cited in the application, and the methods of the published application, including the Sequences of the application, the references cited in the application, the references cited in the application, the references cited in the application of the fields of the application, the application of the references cited in the application, the various references cited in the fields of the application, and the methods of the application of the methods of the invention, the application, and the methods of the application, the application of the application, the methods of the invention, and the application, the methods of the invention, the application, the methods of the invention, and the methods of the invention, and.

Table 1: CDR definition

¹Residue numbering follows the Kabat et al nomenclature described previously

²Residue numbering follows the aforementioned nomenclature of Chothia et al

³Residue numbering follows the nomenclature of MacCallum et al, supra

⁴Residue numbering follows the aforementioned Lefranc et al nomenclature

⁵Residue numbering follows the nomenclature of Honegger and Pl ü ckthun, supra

The term "chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the biological activity of the invention (see U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

The term "semi-synthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.

The term "fully synthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has an immobilized naturally occurring V_H/V_LThe frames are paired up in a pair, and,and not all 6 CDRs of both the heavy and light chains.

"humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired antibody specificity, affinity, and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues not found in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of the variable domains of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, biol.2:593-596 (1992).

"homology" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are "homologous" at that position. The "percent homology" or "percent sequence identity" between two sequences is a function of the number of matching or homologous positions common to the two sequences divided by the number of compared positions multiplied by 100, and any conservative substitutions are considered part of the sequence identity. For example, if 6 of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. In general, a comparison is made when two sequences are aligned to produce the greatest percentage homology. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. One of skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared. However, for purposes herein, the sequence comparison computer program MUSCLE was used to generate% amino acid sequence identity values (Edgar, R.C., Nucleic acids research 32(5):1792-1797, 2004; Edgar, R.C., BMC biologics 5(1):113,2004).

"C" of human IgG Fc region_HThe 1 domain (also referred to as the "C1" domain of "H1") typically extends from about amino acid 118 to about amino acid 215(EU numbering system).

Unless otherwise specified, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. A phrase a nucleotide sequence encoding a protein or RNA may also comprise introns to the extent that the protein-encoding nucleotide sequence may contain introns in some versions.

The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. In general, operably linked DNA sequences are contiguous and, when necessary to join two protein coding regions, in the same reading frame.

The term "inducible promoter" refers to a promoter whose activity can be regulated by the addition or removal of one or more specific signals. For example, an inducible promoter can activate transcription of an operably linked nucleic acid under a particular set of conditions, e.g., in the presence of an inducer or condition that activates the promoter and/or alleviates suppression of the promoter.

As used herein, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include (but are not limited to) one or more of the following: relieving one or more symptoms caused by the disease, lessening the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, ameliorating the disease state, causing disease remission (partial or complete), reducing the dose of one or more other drugs required to treat the disease, delaying disease progression, increasing or improving quality of life, increasing weight gain, and/or prolonging survival. "treating" also encompasses reducing the pathological consequences of a disease (such as tumor volume of cancer). The methods of the invention encompass any one or more of these therapeutic aspects.

The term "recurrence/relapse/relapsed" refers to the recovery of cancer or disease after clinical assessment of disease disappearance. Diagnosis of distant metastasis or local recurrence of cancer can be considered recurrence.

The term "refractory" or "resistant" refers to a cancer or disease that does not respond to treatment.

An "effective amount" of a caTCR, or a composition comprising a caTCR, as disclosed herein is an amount sufficient for the purposes specifically recited. An "effective amount" can be determined empirically and by known methods relevant to the stated purpose.

The term "therapeutically effective amount" refers to an amount of a caTCR, or a composition comprising a caTCR, as disclosed herein that is effective to "treat" an individual disease or disorder. In the case of cancer, a therapeutically effective amount of a caTCR, or a composition comprising a caTCR, as disclosed herein can reduce the number of cancer cells; reducing tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or relieve to some extent one or more symptoms associated with cancer. To the extent that the caTCR, or a composition comprising the caTCR, as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, a therapeutically effective amount is an amount that improves progression-free survival of a patient. In the case of an infectious disease, such as a viral infection, a therapeutically effective amount of a caTCR, or a composition comprising a caTCR, as disclosed herein can reduce the number of cells infected by the pathogen; reducing the production or release of antigens derived from a pathogen; inhibit (i.e., slow to some extent and preferably stop) pathogen diffusion into uninfected cells; and/or relieve to some extent one or more symptoms associated with the infection. In some embodiments, a therapeutically effective amount is an amount that prolongs the survival of a patient.

As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The pharmaceutically acceptable carrier or excipient preferably meets the required standards for toxicological and manufacturing testing and/or is included in the Inactive ingredient guide (Inactive ingredient guide) established by the U.S. food and Drug administration.

It is to be understood that the embodiments of the invention described herein include "consisting of an embodiment" and/or "consisting essentially of an embodiment.

Herein, reference to "about" a value or parameter includes (and describes) variations that are directed to the value or parameter itself. For example, descriptions that refer to "about X" include descriptions of "X".

As used herein, reference to "not" a value or parameter generally means and describes "in addition to" a value or parameter. For example, a method not used to treat type X cancer means that the method is used to treat a type of cancer other than X.

As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

caTCR plus CSR immune cells

The present invention provides an immune cell (such as a T cell) that presents on its surface a caTCR and a CSR according to any of the caTCR and CSR described herein (such immune cell is also referred to herein as a "caTCR plus CSR immune cell"). In some embodiments, the immune cell comprises a nucleic acid encoding a cabTCR and a CSR, wherein the cabTCR and CSR are expressed from the nucleic acid and are localized on the surface of the immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cells are α β T cells and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the T cells are γ δ T cells and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of the endogenous TCR subunits of the immune cell. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. Cell modifications for disrupting gene expression include any such technique known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN-based gene knockout), and the like.

For example, in some embodiments, an immune cell (such as a T cell) is provided that includes a nucleic acid encoding a caTCR according to any of the catcrs described herein and a CSR according to any of the CSRs described herein, wherein the caTCR and CSR are expressed from the nucleic acid and are localized on the surface of the immune cell. In some embodiments, the nucleic acid comprises a first caTCR nucleic acid sequence encoding a first caTCR polypeptide chain of a caTCR, a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of a caTCR, and a CSR nucleic acid sequence encoding a CSR polypeptide chain of a CSR. In some embodiments, the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are each included in a different vector. In some embodiments, some or all of the nucleic acid sequences are included in the same vector. The vector may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors, such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In some embodiments, one or more vectors are integrated into the host genome of the immune cell. In some embodiments, the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are each under the control of different promoters. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

Thus, in some embodiments, there is provided a calcr plus CSR immune cell (such as a T cell) that expresses on its surface a calcr according to any one of the calcrs described herein and a CSR according to any one of the CSRs described herein, wherein the calcr plus CSR immune cell comprises: a) a first caTCR nucleic acid sequence encoding a first caTCR polypeptide chain of a caTCR; b) a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of a caTCR; and c) a CSR nucleic acid sequence encoding a CSR polypeptide chain of a CSR, wherein the first and second caTCR polypeptide chains are expressed from the first and second caTCR nucleic acid sequences to form a caTCR, wherein the CSR polypeptide chain is expressed from the CSR nucleic acid to form a CSR, and wherein the caTCR and the CSR are localized to the surface of an immune cell. In some embodiments, the first caTCR nucleic acid sequence is included in a first vector (such as a lentiviral vector), the second caTCR nucleic acid sequence is included in a second vector (such as a lentiviral vector), and the CSR nucleic acid sequence is included in a third vector (such as a lentiviral vector). In some embodiments, some or all of the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequences are included in the same vector (such as a lentiviral vector). In some embodiments, the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are each individually operably linked to a promoter. In some embodiments, some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors). In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an α β T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the immune cell is a γ δ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors) that integrate into the host genome of the immune cell.

In some embodiments, there is provided a calcr plus CSR immune cell (such as a T cell) that expresses on its surface a calcr according to any one of the calcrs described herein and a CSR according to any one of the CSRs described herein, wherein the calcr plus CSR immune cell comprises: a) a first vector comprising a first promoter operably linked to a first caTCR nucleic acid sequence encoding a first caTCR polypeptide chain of a caTCR; b) a second vector comprising a second promoter operably linked to a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of a caTCR; and c) a third vector comprising a third promoter operably linked to a CSR nucleic acid sequence encoding a CSR polypeptide chain, wherein the first and second caTCR polypeptide chains are expressed by the first and second caTCR nucleic acid sequences to form a caTCR, and the CSR polypeptide chain is expressed by the CSR nucleic acid sequence to form a CSR, and wherein the caTCR and the CSR are localized to the surface of the immune cell. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an α β T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the immune cell is a γ δ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.

In some embodiments, there is provided a calcr plus CSR immune cell (such as a T cell) that expresses on its surface a calcr according to any one of the calcrs described herein and a CSR according to any one of the CSRs described herein, wherein the calcr plus CSR immune cell comprises: a) a first carrier, comprising: i) a first promoter operably linked to the first caTCR nucleic acid sequence of the first caTCR polypeptide chain encoding a caTCR, and ii) a second promoter operably linked to the second caTCR nucleic acid sequence of the second caTCR polypeptide chain encoding a caTCR; and b) a second vector comprising a third promoter operably linked to a CSR nucleic acid sequence encoding a CSR polypeptide chain, wherein the first and second caTCR polypeptide chains are expressed by the first and second caTCR nucleic acid sequences to form a caTCR, and the CSR polypeptide chain is expressed by the CSR nucleic acid sequence to form a CSR, and wherein the caTCR is localized to the surface of an immune cell. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an α β T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the immune cell is a γ δ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell. It is understood that embodiments in which any of the nucleic acid sequences are exchanged, such as the first or second caTCR nucleic acid sequence is exchanged with a CSR nucleic acid sequence, are also contemplated.

In some embodiments, there is provided a calcr plus CSR immune cell (such as a T cell) that expresses on its surface a calcr according to any one of the calcrs described herein and a CSR according to any one of the CSRs described herein, wherein the calcr plus CSR immune cell comprises: a) a first carrier, comprising: i) a first caTCR nucleic acid sequence encoding a first caTCR polypeptide chain of a caTCR, and ii) a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of a caTCR, wherein the first and second caTCR nucleic acid sequences are under the control of a first promoter; and b) a second vector comprising a third promoter operably linked to a CSR nucleic acid sequence encoding a CSR polypeptide chain, wherein the first and second caTCR polypeptide chains are expressed by the first and second caTCR nucleic acid sequences to form a caTCR and the CSR polypeptide chain is expressed by the CSR nucleic acid sequence to form a CSR, and wherein the caTCR and the CSR are localized to the surface of an immune cell. In some embodiments, the first promoter is operably linked to the 5' end of the first catr nucleic acid sequence, and a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) is present to link the 3' end of the first catr nucleic acid sequence to the 5' end of the second catr nucleic acid sequence, wherein the first and second catr nucleic acid sequences are transcribed as a single RNA under the control of the promoter. In some embodiments, the first promoter is operably linked to the 5' end of the second catr nucleic acid sequence, and a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) is present to link the 3' end of the second catr nucleic acid sequence to the 5' end of the first catr nucleic acid sequence, wherein the first and second catr nucleic acid sequences are transcribed as a single RNA under the control of the promoter. In some embodiments, the first and/or second promoters have the same sequence. In some embodiments, the first and/or second promoters have different sequences. In some embodiments, the first and/or second promoter is inducible. In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an α β T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the immune cell is a γ δ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of an immune cell. It is understood that embodiments in which any of the nucleic acid sequences are exchanged, such as the first or second caTCR nucleic acid sequence is exchanged with a CSR nucleic acid sequence, are also contemplated.

In some embodiments, there is provided a calcr plus CSR immune cell (such as a T cell) that expresses on its surface a calcr according to any one of the calcrs described herein and a CSR according to any one of the CSRs described herein, wherein the calcr plus CSR immune cell comprises a vector comprising a) a first calcr nucleic acid sequence encoding a first calcr polypeptide chain of a calcr; b) a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of a caTCR; and c) a CSR nucleic acid sequence encoding a CSR polypeptide chain of a CSR, wherein the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are under the control of a single promoter; and wherein the first and second caTCR polypeptide chains are expressed from the first and second caTCR nucleic acid sequences to form a caTCR and the CSR polypeptide chain is expressed from the CSR nucleic acid sequence to form a CSR, and wherein the caTCR and the CSR are localized to the surface of the immune cell. In some embodiments, the promoter is operably linked to one of the nucleic acid sequences linked to the other nucleic acid sequence by a nucleic acid linker individually selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide, such as P2A, T2A, E2A, or F2A, such that the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are transcribed into a single RNA under the control of the promoter. In some embodiments, the promoter is inducible. In some embodiments, the immune cell does not express a TCR subunit from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an α β T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR δ and γ chains, or the immune cell is a γ δ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains, or the immune cell is a γ δ T cell modified to block or reduce expression of TCR γ and/or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of an immune cell.

Chimeric antibody/T cell receptor (caTCR) constructs

In one aspect, a target antigen-specific chimeric antibody/T cell receptor (caTCR) described herein specifically binds to a target antigen (such as a cell surface antigen or a peptide/MHC complex) and is capable of recruiting at least one TCR-associated signaling molecule (such as CD3 δ ∈, CD3 γ ∈, and/or ζ ζ). In some embodiments, the anti-caTCR comprises a naturally occurring TCR domain. In some embodiments, the caTCR comprises at least one non-naturally occurring TCR domain. The caTCR includes an antigen binding module that provides antigen specificity and a T Cell Receptor Module (TCRM) that allows for CD3 recruitment and signaling. The antigen binding moiety is not a naturally occurring T cell receptor antigen binding moiety. In some embodiments, the antigen binding module is linked to the amino terminus of a polypeptide chain in the TCRM. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific). TCRMs include transmembrane modules derived from the transmembrane domain of one or more TCRs (TCR-TM), such as α β and/or γ δ TCRs, and optionally further include one or both of a connecting peptide of a TCR, or a fragment thereof, and/or one or more TCR endodomains, or a fragment thereof. In some embodiments, the TCRM comprises two polypeptide chains, each polypeptide chain comprising, from amino-terminus to carboxy-terminus, a connecting peptide, a transmembrane domain, and optionally a TCR endodomain. In some embodiments, the TCRM comprises one or more non-naturally occurring TCR domains. For example, in some embodiments, the TCRM comprises one or two non-naturally occurring TCR transmembrane domains. A non-naturally occurring TCR domain may be the corresponding domain of a naturally occurring TCR, modified by substitution of one or more amino acids and/or by replacing a portion of the corresponding domain with a portion of an analogous domain from another TCR. The caTCR can comprise a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form an antigen binding module and a TCRM. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the caTCR is a multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide bond or by another chemical bond (such as a disulfide bond). In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond. In some embodiments, the caTCR further comprises one or more T cell costimulatory signaling sequences. The one or more costimulatory signaling sequences can individually be all or a portion of the intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, one or more co-stimulatory signaling sequences are between the first TCR-TM and the first TCR endodomain and/or between the second TCR-TM and the second TCR endodomain. In some embodiments, one or more co-stimulatory signaling sequences are at the carboxy terminus of the first TCRD and/or the second TCRD. In some embodiments, the caTCR lacks a T cell costimulatory signaling sequence. In some embodiments, the caTCR further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the caTCR. In some embodiments, the stabilizing moiety is located between the antigen binding moiety and the TCRM. In some embodiments, the caTCR further comprises a spacer module between any two caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers connecting two caTCR modules or domains.

The caTCR described herein can have one or more features described in this section. It is intended that any of the features of the components of the cadRs described herein (e.g., antigen binding module, TCRD, TCR-TM, spacer module, stabilizing module, T cell co-stimulatory sequences and various linkers, etc.) can be combined with each other, with any of the features of CSR, and with any of the features of SSE, as if each combination were individually described.

In some embodiments, the antigen binding moiety (such as an antibody moiety) has the following affinity or K_dSpecific binding to a target antigen: a) the affinity is at least about 10-fold (including, e.g., at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000, or more fold) greater than its binding affinity for other molecules; b) said K_dNot exceeding K bound to other molecules_dAbout 1/10 (such as no more than any of about 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000, or less). Binding affinity can be determined by methods known in the art, such as ELISA, Fluorescence Activated Cell Sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). K_dCan be determined by methods known in the art, such as Surface Plasmon Resonance (SPR) analysis using, for example, a Biacore instrument, or kinetic exclusion analysis using, for example, a Sapidyne instrument (KinExA).

Examples of a stability domain include an Fc region; a hinge region; c _H3 domains; c _H4 domains; c _H1 or C_LA domain; leucine zipper domain (e.g., jun/fos leucine zipper domain, see, e.g., Kostelney et al, J.Immunol,148: 1547-; an isoleucine zipper domain; dimeric regions of dimeric cell surface receptors (e.g., interleukin-8 receptor (IL-8R) or integrin heterodimers (such as LFA-1 or GPIIIb/IIIa), secretory dimeric ligands (e.g., Nerve Growth Factor (NGF), neurotrophin-3 (NT-3), interleukin-8 (IL-8)) Vascular Endothelial Growth Factor (VEGF) or Brain Derived Neurotrophic Factor (BDNF); see, e.g., Arakawa et al, J.biol.chem.269:27833-27839,1994, and Radziewski et al, biochem.32:1350,1993); coiled-coil dimerization domains (see, e.g., WO 2014152878; Fletcher et al, ACS Synth. biol.1: 240-; and a polypeptide comprising at least one cysteine residue (e.g., about one, two, or three to about ten cysteine residues) such that a disulfide bond can be formed between the polypeptide and a second polypeptide comprising at least one cysteine residue.

In some embodiments, the TCRM described herein comprises: a) a first T Cell Receptor Domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM); and b) a second TCRD comprising a second TCR-TM, wherein the TCRM contributes to recruitment of at least one TCR-related signaling molecule. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, both TCR-TMs are non-naturally occurring. In some embodiments, the first TCR-TM is derived from one of the transmembrane domains of a T cell receptor (such as an α β TCR or a γ δ TCR), and the second TCR-TM is derived from another transmembrane domain of the T cell receptor. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a transmembrane domain of a naturally occurring T cell receptor. Recruitment of TCR-associated signaling molecules can be determined by methods known in the art, such as FACS analysis of surface expression of the TCR-CD3 complex or co-immunoprecipitation of the CD3 subunit with catrs.

For example, in some embodiments, a first TCR-TM of a TCRM described herein comprises, consists essentially of, or consists of a transmembrane domain of a TCR α chain (e.g., GenBank accession number: CCI73895) or a variant thereof; and a second TCR-TM of the TCRM comprises, consists essentially of, or consists of a transmembrane domain of a TCR β chain (e.g., GenBank accession number: CCI73893) or a variant thereof. In some embodiments, the first TCR-TM comprises, consists essentially of, or consists of a transmembrane domain of a TCR delta chain (e.g., Genbank accession number: AAQ57272) or a variant thereof; and a second TCR-TM comprises, consists essentially of, or consists of a transmembrane domain of a TCR γ chain (e.g., Genbank accession number: AGE91788) or a variant thereof. In some embodiments, the first and second TCR-TMs of the TCRM described herein comprise, consist essentially of, or consist of, respectively, the following transmembrane domains: the transmembrane domain of the constant domain of TCR alpha chain (e.g. SEQ ID NO:1) or a variant thereof, and the transmembrane domain of the constant domain of TCR beta chain (e.g. SEQ ID NO:2) or a variant thereof. In some embodiments, the first and second TCR-TMs each comprise, consist essentially of, or consist of the following transmembrane domains: the transmembrane domain of the TCR delta chain constant domain (e.g., SEQ ID NO:3) or a variant thereof, and the transmembrane domain of the TCR gamma chain constant domain (e.g., SEQ ID NO:4) or a variant thereof. In some embodiments, the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOS 5 and 6, respectively, or variants thereof. In some embodiments, the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs 7 and 8, respectively, or variants thereof. Variants of a transmembrane domain include, but are not limited to, transmembrane domains with one or more amino acid substitutions as compared to a reference sequence. In some embodiments, the variant transmembrane domain comprises no more than 5 amino acid substitutions compared to the reference sequence. In some embodiments, the first TCRD further comprises a first linking peptide at the amino terminus of the transmembrane domain and/or the second TCRD further comprises a second linking peptide at the amino terminus of the transmembrane domain. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and/or second connecting peptide comprises, consists essentially of, or consists of, respectively: all or a portion of the connecting peptide of the TCR alpha chain constant domain (e.g., SEQ ID NO:1) or a variant thereof, and all or a portion of the connecting peptide of the TCR beta chain constant domain (e.g., SEQ ID NO:2) or a variant thereof. In some embodiments, the first and/or second connecting peptide comprises, consists essentially of, or consists of, respectively: all or a portion of the linker peptide of SEQ ID NO 27 or 28 or a variant thereof, and all or a portion of the linker peptide of SEQ ID NO 29 or 30 or a variant thereof. In some embodiments, the first and/or second connecting peptide comprises, consists essentially of, or consists of, respectively: all or a portion of the connecting peptide of the TCR delta chain constant domain (e.g., SEQ ID NO:3) or a variant thereof, and all or a portion of the connecting peptide of the TCR gamma chain constant domain (e.g., SEQ ID NO:4) or a variant thereof. In some embodiments, the first and/or second connecting peptide comprises, consists essentially of, or consists of, respectively: all or a portion of the linker peptide of SEQ ID NO 31 or 32 or a variant thereof, and all or a portion of the linker peptide of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first TCRD further comprises a first TCR endodomain at the carboxy terminus of the first TCR-TM, and/or the second TCRD further comprises a second TCR endodomain at the carboxy terminus of the second TCR-TM. In some embodiments, the first TCR endodomain comprises all or a portion of, or a variant thereof, an endodomain of a TCR subunit from which the first TCR-TM is derived, and/or the second endodomain comprises all or a portion of, or a variant thereof, an endodomain of a TCR subunit from which the second TCR-TM is derived. In some embodiments, the second TCR intracellular domain comprises any one of the amino acid sequences of SEQ ID NOS 35-36 or a variant thereof. In some embodiments, the first TCRD is a fragment of one chain of a naturally occurring TCR, or a variant thereof, and/or the second TCRD is a fragment of another chain of a naturally occurring TCR, or a variant thereof. In some embodiments, at least one of the TCRDs is non-naturally occurring. In some embodiments, the first and second TCRDs are linked by a disulfide bond. In some embodiments, the first and second TCRDs are linked by a disulfide bond between a residue in the first linking peptide and a residue in the second linking peptide. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM is capable of recruiting each of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ to form the catr-CD 3 complex (i.e., promote the formation of the catr-CD 3 complex).

Contemplated caTCR constructs include, for example, caTCR that specifically binds to a cell surface antigen, caTCR that specifically binds to a cell surface presenting peptide/MHC complex, and caTCR that specifically binds to both a cell surface antigen and a cell surface presenting peptide/MHC complex.

In some embodiments, the antigen binding moiety is an antibody moiety selected from the group consisting of: fab, Fab ', (Fab')2, Fv or single chain Fv (scFv). In some embodiments, the antibody moiety is monospecific. In some embodiments, the antibody moiety is multispecific. In some embodiments, the antibody moiety is bispecific. In some embodiments, the antibody moiety is a tandem scFv, a bifunctional antibody (Db), a single chain bifunctional antibody (scDb), a parental force retargeting (DART) antibody, a Double Variable Domain (DVD) antibody, a chemically cross-linked antibody, a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the antibody moiety is a tandem scFv comprising two scfvs linked by a peptide linker. In some embodiments, the antibody moiety is two scfvs that are not directly linked. In some embodiments, the antibody moiety is a fully human antibody moiety, a semi-synthetic antibody moiety having human antibody framework regions, or a humanized antibody moiety.

In some embodiments, the antigen binding moiety comprises a V-containing moiety_HFirst antigen binding domain of antibody domain and V-containing_LA second antigen-binding domain of the antibody domain. In some embodiments, all V_HAntibody domains and V_LThe antibody domain CDRs are all derived from the same antibody portion. In some embodiments, some V_HAntibody domains and V_LThe antibody domain CDRs are all derived from different antibody portions. In some embodiments, V_HAntibody domains and/or V_LThe antibody domain is a human, humanized, chimeric, semi-synthetic, or fully synthetic antibody domain.

In some embodiments, the antigen-binding moiety is a semi-synthetic antibody moiety comprising a fully human sequence and one or more synthetic regions. In some embodiments, the antigen binding moiety is a peptide comprising a fully human V_LAnd semi-synthesis of V_HThe semi-synthetic antibody portion of (1), which comprises the fully human FR1, HC-CDR1, FR2, HC-CDR2, FR3 and FR4 regions and the synthetic HC-CDR 3. In some embodiments, semisynthetic V_HIncluding the fully synthetic HC-CDR3,the sequence is about 5 to about 25 (such as any of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length. In some embodiments, semisynthetic V_HOr the synthetic HC-CDR3 is obtained from a semisynthetic library (such as a semisynthetic human library) comprising a fully synthetic HC-CDR3 region having a sequence length of about 5 to about 25 (such as any of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, wherein each amino acid in the sequence is randomly selected, independently of the other, from a standard human amino acid minus cysteine. In some embodiments, the synthetic HC-CDR3 is about 10 to about 19 (such as any of about 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acids in length. In some embodiments, the antigen binding moiety is a semi-synthetic antibody moiety, including semi-synthetic V_LAnd semi-synthesis of V_H. In some embodiments, the antigen binding moiety is a peptide comprising human V with an immobilization_H/V_LFramework paired fully synthetic antibody portions, but all 6 CDRs of the heavy and light chains were randomly synthesized sequences.

In some embodiments, the antigen binding moiety is an antibody moiety comprising specific CDR sequences derived from one or more antibody moieties (such as monoclonal antibodies) or certain variants of such sequences comprising one or more amino acid substitutions. In some embodiments, the amino acid substitutions in the variant sequence do not substantially reduce the ability of the antigen-binding moiety to bind to the target antigen. Changes that substantially improve the binding affinity of the target antigen or affect some other property, such as specificity and/or cross-reactivity with related variants of the target antigen, are also contemplated.

In some embodiments, the stabilizing moiety is derived from an antibody moiety. For example, in some embodiments, the stabilization module comprises C _H1 an antibody domain or variant thereof, and a polypeptide comprising a first stabilizing domain_LA second stabilizing domain of an antibody domain or variant thereof. In another embodiment, the stabilization module comprises C_H3a first stabilizing domain of an antibody domain or variant thereof, and a polypeptide comprising C_H3a second stabilizing domain of an antibody domain or variant thereof. In some embodiments, the antibody heavy chain constant domain (e.g., C) contained in the stabilizing moiety _H1 or C_H3) Derived from an IgG (e.g. IgG1, IgG2, IgG3 or IgG4), IgA (e.g. IgA1 or IgA2), IgD, IgM or IgE heavy chain, optionally human. In some embodiments, the antibody heavy chain constant domain (e.g., C) contained in the stabilizing moiety _H1 or C_H3) Variants that include one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) as compared to the sequence from which the antibody heavy chain constant domain is derived. In some embodiments, the antibody light chain constant domain (C) contained in the stabilizing moiety_L) Derived from a kappa or lambda light chain, optionally human. In some embodiments, the antibody light chain constant domain (C) contained in the stabilizing moiety_L) Variants that include one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) as compared to the sequence from which the constant domain of the light chain of the antibody is derived. In some embodiments, the first and/or second stabilizing domains comprise one or more modifications that do not substantially alter their binding affinity for each other. In some embodiments, the first and/or second stabilizing domain comprises one or more modifications that increase their binding affinity to each other and/or introduce non-naturally occurring disulfide bonds. In some embodiments, the stabilization module comprises knob-and-hole (see, e.g., Carter P.J Immunol methods.248:7-15,2001) modifications. For example, in some embodiments, the stabilizing moiety comprises an antibody constant domain region (e.g., C) comprising a knob-hole modification _H3 fields). In some embodiments, the stabilizing moiety comprises an antibody constant domain region (e.g., C) modified by electrostatic steering to enhance binding thereof_H3 domains) (see, e.g., WO2006106905 and Gunasekaran K et al J Biol chem.285:19637-46, 2010). In some embodiments, the first and second stabilizing domains are linked by a disulfide bond.

In some embodiments, the caTCR comprises an antigen binding moiety described herein linked to a TCRM described herein, optionally comprising a stabilizing moiety. For example, in some embodiments, the caTCR includes an antigen binding moiety attached to the N-terminus of one or both of the TCRDs. In some embodiments, the caTCR comprises a stabilizing moiety between the TCRM and the antigen binding moiety. In some embodiments, the caTCR further comprises a spacer module between any two caTCR modules or domains. In some embodiments, the spacer moiety comprises one or more peptide linkers between about 5 to about 70 (such as any of about 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any range between these values) amino acids in length. In some embodiments, the caTCR further comprises one or more accessory intracellular domains. In some embodiments, the one or more auxiliary endodomains are carboxy-terminal to the first and/or second TCRD. In some embodiments, the one or more accessory endodomains are between a first TCR-TM and a first TCR endodomain, and/or between a second TCR-TM and a second TCR endodomain. In some embodiments, the one or more accessory intracellular domains individually comprise a TCR costimulatory domain. In some embodiments, the TCR costimulatory domain comprises all or a portion of the intracellular domain of an immune costimulatory molecule, such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the TCR co-stimulatory domain comprises all or a portion of the amino acid sequence of any of SEQ ID NOS 51-56 or a variant thereof.

In some embodiments, the antigen binding moiety specifically binds to a cell surface antigen. In some embodiments, the cell surface antigen is selected from the group consisting of proteins, carbohydrates, and lipids. In some embodiments, the cell surface antigen is a disease-associated antigen expressed in a diseased cell. In some embodiments, the antigen binding moiety specifically binds to a complex comprising a peptide and an MHC protein. peptide/MHC complexes include, for example, surface presentation complexes including peptides derived from disease-associated antigens expressed in diseased cells and MHC proteins. In some embodiments, the full-length disease-associated antigen is not generally expressed on the surface of the diseased cell (e.g., the disease-associated antigen is an intracellular or secreted protein). In some embodiments, the disease is cancer and the disease-associated antigen is a tumor-associated antigen expressed in cancer cells. In some embodiments, the tumor-associated antigen is an oncoprotein. In some embodiments, the cancerThe protein is the result of a mutation in the proto-oncogene and the oncoprotein includes a neoepitope containing the mutation. For example, in some embodiments, the antigen-binding moiety specifically binds to a cell surface tumor-associated antigen (e.g., an oncoprotein that includes a neoepitope). In some embodiments, the antigen binding module specifically binds to a complex comprising a peptide derived from a tumor-associated antigen (e.g., an oncoprotein comprising a neoepitope) that is not normally expressed on the surface of a cancer cell (e.g., an intracellular or secreted tumor-associated antigen) and an MHC protein. In some embodiments, the disease is a viral infection and the disease-associated antigen is a virus-associated antigen expressed in infected cells. For example, in some embodiments, the antigen binding moiety specifically binds to a cell surface virus-associated antigen. In some embodiments, the antigen binding module specifically binds to a complex comprising a peptide derived from a virus-associated antigen that is not normally expressed on the surface of a virus-infected cell (e.g., intracellular or secreted virus-associated antigen) and an MHC protein. In some embodiments, the caTCR construct has a K between about 0.1pM to about 500nM (such as any of about 0.1pM, 1.0pM, 10pM, 50pM, 100pM, 500pM, 1nM, 10nM, 50nM, 100nM or 500nM, including any range between these values)_dBinding to the target antigen.

In some embodiments, the caTCR comprises an antigen binding moiety that specifically binds to a cell surface antigen, wherein the cell surface antigen is CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL5, including variants or mutants thereof. Specific binding to a complete antigen, such as a cell surface antigen, is sometimes referred to as "non-MHC restricted binding".

In some embodiments, the caTCR comprises an antigen binding moiety that specifically binds to a complex comprising a peptide and an MHC protein, wherein the peptide is derived from a protein selected from the group consisting of: WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. Specific binding to a complex comprising a peptide and an MHC protein is sometimes referred to as "MHC restricted binding".

In some embodiments, the caTCR includes an antigen binding moiety that specifically binds to a complex comprising a peptide derived from a disease-associated antigen (such as a tumor-associated or virus-encoded antigen) and an MHC class I protein, wherein the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. In some embodiments, the MHC class I protein is HLA-A, HLA-B or HLA-C. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein is HLA-B. In some embodiments, the MHC class I protein is HLA-C. In some embodiments, the MHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is any one of HLA-A02: 01-555, such as HLA-A02: 01, HLA-A02: 02, HLA-A02: 03, HLA-A02: 04, HLA-A02: 05, HLA-A02: 06, HLA-A02: 07, HLA-A02: 08, HLA-A02: 09, HLA-A02: 10, HLA-A02: 11, HLA-A02: 12, HLA-A02: 13, HLA-A02: 14, HLA-A02: 15, HLA-A02: 16, HLA-A02: 17, HLA-A02: 18, HLA-A02: 19, HLA-A02: 20, HLA-A02: 21, HLA-A02: 22, or HLA-A24. In some embodiments, the MHC class I protein is HLA-a 02: 01.

In some embodiments, the caTCR includes an antigen binding moiety that specifically binds to a complex comprising a peptide derived from a disease-associated antigen (such as a tumor-associated or virus-encoded antigen) and an MHC class II protein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, the MHC class II protein is HLA-DP. In some embodiments, the MHC class II protein is HLA-DQ. In some embodiments, the MHC class II protein is HLA-DR.

In some embodiments, a caTCR described herein comprises: a) an antigen binding moiety that specifically binds to a target antigen, and b) a TCRM comprising first and second TCR-TMs derived from transmembrane domains of a TCR (such as an α β TCR or a γ δ TCR), wherein the TCRM is capable of recruiting at least one TCR-associated signaling molecule. In some embodiments, the antigen binding moiety is linked to one or more of the TCRMsThe amino terminus of the polypeptide chain. For example, in some embodiments, the TCRM comprises two polypeptide chains, and the antigen binding module is connected to the amino terminus of one or both of the TCRM polypeptide chains. In some embodiments, the first and second TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first and second TCR-TMs are non-naturally occurring. In some embodiments, the TCRM further comprises at least one connecting peptide of a TCR, or a fragment thereof, at the amino terminus of the TCR-TM. In some embodiments, the TCRM further comprises at least one TCR endodomain comprising a sequence from the intracellular domain of a TCR at the carboxy terminus of the TCR-TM. In some embodiments, the TCRM comprises a TCRD derived from a fragment of a TCR chain. In some embodiments, at least one of the TCRDs is non-naturally occurring. In some embodiments, the caTCR further comprises at least one accessory intracellular domain comprising a T cell costimulatory signaling sequence at the carboxy terminus of the TCR-TM (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the caTCR lacks a costimulatory signaling sequence. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety comprises V_HAntibody domains and V_LAn antibody domain. In some embodiments, the antibody moiety is human, humanized, chimeric, semi-synthetic, or fully synthetic. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the stabilizing moiety comprises at least one disulfide bond linking the stabilizing domains. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodimentsIn (e), a spacer moiety is present between any two caTCR moieties or domains. In some embodiments, the caTCR is a heteromultimer, such as a heterodimer. For example, in some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a first TCRD and a second polypeptide chain comprising a second TCRD, wherein the antigen binding moiety is linked to the first and/or second polypeptide chain. In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the TCR is an α β TCR, and the first and second TCR-TMs are derived from TCR α and β subunit transmembrane domains. In some embodiments, the TCR is a γ δ TCR, and the first and second TCR-TMs are derived from TCR γ and δ subunit transmembrane domains. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain comprises the sequence of an endodomain from a TCR subunit from which the first TCR-TM is derived, and/or the second TCR endodomain comprises the sequence of an endodomain from a TCR subunit from which the second TCR-TM is derivedAnd (4) columns. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring α β TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring α β TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In thatIn some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the first TCR-TM is derived, and/or the second TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a naturally occurring α β T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments of the present invention, the,the antibody moiety is Fab, Fab ', (Fab')2, Fv or single chain Fv (scFv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring γ δ TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring γ δ TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the first TCR-TM is derived, and/or the second TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domainTwo stabilizing domains, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the caTCR. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a naturally occurring γ δ T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO 1 and SEQ ID NO 2; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of one another, comprise the amino acid sequence of a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first and second linking peptides are each otherThis independently includes the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of an endodomain contained in any one of SEQ ID NOs 1-4, or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 1 and 2. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising the sequences derived from SEQ ID NO 1 and2, a first TCR-TM of a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein at least one of the TCR-TM comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions as compared to the amino acid sequence from which the TCR-TM is derived, and the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of each other comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions compared to the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of one another, comprise the amino acid sequence of a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR intracellular domain and the second TCR intracellular domain each independently of the other comprise any of SEQ ID NOS: 1-4An amino acid sequence of an intracellular domain contained in one of the above-mentioned amino acid sequences or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 1 and 2. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO 1 and SEQ ID NO 2; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein at least one of the TCR-TMs comprises a chimeric sequence comprising a portion of contiguous amino acids from the transmembrane domain contained in SEQ ID NO 3 or 4, and the first and second TCRDs form a T capable of recruiting at least one TCR-related signaling moleculeCRM; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of the other comprises a chimeric sequence comprising a portion of contiguous amino acids from the transmembrane domain contained in SEQ ID NO 3 or 4. In some embodiments, the first TCR-TM and/or the second TCR-TM each independently of the other comprise a chimeric sequence comprising NO more than about 10 (such as NO more than about 9, 8, 7, 6,5 or less) contiguous amino acids from a portion of the transmembrane domain contained in SEQ ID NO:3 or 4. In some embodiments, the chimeric sequence in the first or second TCR-TM is from the transmembrane domain contained in SEQ ID NO 3 and the chimeric sequence in the other TCR-TM is from the transmembrane domain contained in SEQ ID NO 4. In some embodiments, the chimeric sequence in the first TCR-TM is positioned such that it can interact with the chimeric sequence in the second TCR-TM. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of one another, comprise the amino acid sequence of a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of an endodomain contained in any one of SEQ ID NOs 1-4, or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the caTCR further comprisesA stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the caTCR. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 1 and 2. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO 3 and SEQ ID NO 4; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, each of the first and second linking peptides independently of the other comprises a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4An amino acid sequence or a variant thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of an endodomain contained in any one of SEQ ID NOs 1-4, or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 3 and 4. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, described hereinThe caTCR of (1) that specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO 3 and SEQ ID NO 4; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein at least one of the TCR-TM comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions as compared to the amino acid sequence from which it is derived, and the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of each other comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions compared to the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of one another, comprise the amino acid sequence of a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodimentsThe first and second TCR intracellular domains each independently of the other comprise the amino acid sequence of the intracellular domain contained in any one of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 3 and 4. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from a transmembrane domain contained in one of the amino acid sequences of SEQ ID NO 31 and SEQ ID NO 4; and a second TCRD comprising a second TCR-TM derived from a transmembrane domain contained in another amino acid sequence, wherein at least one of the TCR-TMs comprises a chimeric comprising a portion of contiguous amino acids from the transmembrane domain contained in SEQ ID NO 1 or2And the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of the other comprises a chimeric sequence comprising a portion of contiguous amino acids from the transmembrane domain contained in SEQ ID NO 1 or 2. In some embodiments, the first TCR-TM and/or the second TCR-TM each independently of the other comprise a chimeric sequence comprising NO more than about 10 (such as NO more than about 9, 8, 7, 6,5 or less) contiguous amino acids from a portion of the transmembrane domain contained in SEQ ID NO:1 or 2. In some embodiments, the chimeric sequence in the first or second TCR-TM is from the transmembrane domain contained in SEQ ID NO 1 and the chimeric sequence in the other TCR-TM is from the transmembrane domain contained in SEQ ID NO 2. In some embodiments, the chimeric sequence in the first TCR-TM is positioned such that it can interact with the chimeric sequence in the second TCR-TM. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of one another, comprise the amino acid sequence of a linking peptide contained in any one of the amino acid sequences of SEQ ID NOs 1-4 or a variant thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of an endodomain contained in any one of SEQ ID NOs 1-4, or a variant thereof. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the catrs further comprise at least one accessory intracellular domain comprising a T cell costimulatory signaling sequence (such as from a T cell costimulatory signaling sequence)CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of the transmembrane domains contained in SEQ ID NOs 3 and 4. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 5 and SEQ ID NO 6; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second linking peptides each, independently of the other, comprise a peptide of SEQ ID NOS 27-34Any one of the amino acid sequences or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ id nos 5 and 6. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 5 and SEQ ID NO 6; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein at least one of the TCR-TMs comprises one or more (such as 2, 3, 4, 2, 4, b, c) compared to the amino acid sequence from which it is derived,5 or more) amino acid substitutions, and the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of each other comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions compared to the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stability domains are connected by a secondAnd (4) sulfur bond connection. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 5 and 6. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 5 and SEQ ID NO 6; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein at least one of the TCR-TMs comprises a chimeric sequence comprising a portion of contiguous amino acids from SEQ ID NO 7 or 8, and the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of the other comprises a chimeric sequence comprising a portion of contiguous amino acids from SEQ ID NO 7 or 8. In some embodiments, the first TCR-TM and/or the second TCR-TM each independently of the other comprise a chimeric sequence comprising a portion of NO more than about 10 (such as NO more than about 9, 8, 7, 6,5 or less) contiguous amino acids from SEQ ID NO 7 or 8. In some embodiments, the chimeric sequence in the first or second TCR-TM is from SEQ ID NO 7 and the chimeric sequence in the other TCR-TM is from SEQ ID NO 8. In some embodiments, the chimeric sequence in a first TCR-TM is positioned such that it can be aligned with the chimeric sequence in a second TCR-TMAnd (4) interaction. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 5 and 6. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) in the first of the TCRDs, the TCRD,comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 8; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ id nos 7 and 8. In some embodimentsTCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 8; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein at least one of the TCR-TM comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions as compared to the amino acid sequence from which it is derived, and the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of each other comprises one or more (such as 2, 3, 4, 5 or more) amino acid substitutions compared to the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodimentsThe first and second linker peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the amino acid sequences of SEQ ID NO 7 and SEQ ID NO 8; and a second TCRD comprising a second TCR-TM derived from another amino acid sequence, wherein at least one of the TCR-TMs comprises a chimeric sequence comprising a portion of contiguous amino acids from SEQ ID NO 5 or 6, and the first and second TCRD form a loop capable of recruiting at least one TCR phaseTCRM off signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, each of the TCR-TMs independently of the other comprises a chimeric sequence comprising a portion of contiguous amino acids from SEQ ID NO 5 or 6. In some embodiments, the first TCR-TM and/or the second TCR-TM each independently of the other comprise a chimeric sequence comprising a portion of NO more than about 10 (such as NO more than about 9, 8, 7, 6,5 or less) contiguous amino acids from SEQ ID NO 5 or 6. In some embodiments, the chimeric sequence in the first or second TCR-TM is from SEQ ID NO 5 and the chimeric sequence in the other TCR-TM is from SEQ ID NO 6. In some embodiments, the chimeric sequence in the first TCR-TM is positioned such that it can interact with the chimeric sequence in the second TCR-TM. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first and second connecting peptides each, independently of one another, comprise the amino acid sequence of any one of SEQ ID NOs 27-34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain and the second TCR endodomain each, independently of one another, comprise the amino acid sequence of SEQ ID NO 35 or 36, or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one agent selected fromA TCR-associated signaling molecule consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (such as bispecific).

Various aspects are discussed in more detail in the sections below.

TCR-TM variants

In some embodiments, the TCR-TMs of the caTCR are derived from a T cell receptor, wherein at least one of the TCR-TMs is non-naturally occurring. Non-naturally occurring TCR-TMs derived from T cell receptors comprise a transmembrane domain from the T cell receptor that has been modified by substitution of one or more amino acids. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent (either in primary sequence or spatially) to an amino acid in the TCRM that is involved in binding to CD 3. For example, in some embodiments, at least one of the substituted amino acids is separated from the amino acid in TCRM that is involved in binding to CD3 by no more than 3 (such as 0,1, 2, or 3) amino acids. In some embodiments, at least one of the substituted amino acids is separated from the amino acid in TCRM that is involved in binding to CD3 by no more than about 15 (such as no more than about any of 14, 12, 10, 8, 6, 4, 2, or 1) angstroms. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

For example, in some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor comprises, consists essentially of, or consists of: the transmembrane domain of an α, β, γ or δ TCR subunit is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the TCR subunit is modified by a substitution of no more than 5 amino acid residues. In some embodiments, the transmembrane domain of the TCR subunit is modified by substitution of a single amino acid residue. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

Thus, in some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: a transmembrane domain of an alpha TCR subunit comprising the amino acid sequence of the transmembrane domain contained in SEQ ID NO:1 (e.g., SEQ ID NO:5), which transmembrane domain is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the α TCR subunit is modified by substitution of NO more than 5 amino acid residues in the transmembrane domain contained in SEQ ID No. 1. In some embodiments, the transmembrane domain of the α TCR subunit is modified by substitution of a single amino acid residue in the transmembrane domain contained in SEQ ID No. 1. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: a transmembrane domain of an α TCR subunit comprising the amino acid sequence of SEQ ID No. 5, which transmembrane domain is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the α TCR subunit is modified by substitution of NO more than 5 amino acid residues in SEQ ID No. 5. In some embodiments, the transmembrane domain of the α TCR subunit is modified by substitution of a single amino acid residue in SEQ ID No. 5. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: a transmembrane domain of a beta TCR subunit comprising the amino acid sequence of the transmembrane domain contained in SEQ ID NO:2 (e.g., SEQ ID NO:6), which transmembrane domain is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the β TCR subunit is modified by substitution of NO more than 5 amino acid residues in the transmembrane domain contained in SEQ ID No. 2. In some embodiments, the transmembrane domain of the β TCR subunit is modified by a single amino acid residue substitution in the transmembrane domain contained in SEQ ID No. 2. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: a transmembrane domain of a beta TCR subunit comprising the amino acid sequence of SEQ ID NO 6, which transmembrane domain is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the β TCR subunit is modified by substitution of NO more than 5 amino acid residues in SEQ ID No. 6. In some embodiments, the transmembrane domain of the β TCR subunit is modified by substitution of a single amino acid residue in SEQ ID NO 6. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: a transmembrane domain comprising the delta TCR subunit of the amino acid sequence of the transmembrane domain contained in SEQ ID NO 3 (e.g., SEQ ID NO 7), which transmembrane domain is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the delta TCR subunit is modified by substitution of NO more than 5 amino acid residues in SEQ ID No. 3. In some embodiments, the transmembrane domain of the delta TCR subunit is modified by substitution of a single amino acid residue in SEQ ID NO 3. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO:3 (e.g., SEQ ID NO:7) modified by one or more substitutions (such as by a more hydrophobic residue) of the amino acids corresponding to SEQ ID NO: 7: l4, M6, V12, N15, F245 and L25. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO 3 modified by substitutions in the amino acids corresponding to V12 and N15 in SEQ ID NO 7 (such as with more hydrophobic residues). In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID No. 3, modified by one or more substitutions corresponding to the following substitutions in SEQ ID No. 7: L4C, M6V, V12F, N15S, F245S and L25S. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO 3 modified by substitutions corresponding to the V12F and N15S substitutions in SEQ ID NO 7. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of any one of SEQ ID NOs 9-13.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: the transmembrane domain of the delta TCR subunit comprising the amino acid sequence of SEQ ID NO 7, modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the delta TCR subunit is modified by substitution of NO more than 5 amino acid residues in seq id No. 7. In some embodiments, the transmembrane domain of the delta TCR subunit is modified by substitution of a single amino acid residue in SEQ ID NO 7. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID No. 7 modified by one or more substitutions (such as with a more hydrophobic residue) of amino acids corresponding to the following amino acids in SEQ ID No. 7: l4, M6, V12, N15, F245 and L25. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO:7 modified by substitutions in the amino acids corresponding to V12 and N15 in SEQ ID NO:7 (such as with a more hydrophobic residue). In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID No. 7 modified by one or more substitutions corresponding to the following substitutions in SEQ ID No. 7: L4C, M6V, V12F, N15S, F245S and L25S. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO. 7 modified by substitutions corresponding to V12F and N15S in SEQ ID NO. 7. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of any one of SEQ ID NOs 9-13.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: the transmembrane domain of the gamma TCR subunit comprising the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 (e.g., SEQ ID NO:8) is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the gamma TCR subunit is modified by substitution of NO more than 5 amino acid residues in the transmembrane domain contained in SEQ ID No. 4. In some embodiments, the transmembrane domain of the gamma TCR subunit is modified by substitution of a single amino acid residue in the transmembrane domain contained in SEQ ID No. 4. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO:4 (e.g., SEQ ID NO:8) modified by one or more substitutions (such as with a more hydrophobic residue) in the amino acids corresponding to the following amino acids in SEQ ID NO: 8: y1, Y2, M3, L5, L8, V12, V13, F15, a16, I18, C19, C20 and C21. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 modified by substitutions in the amino acids corresponding to Y2, M3, A16 and I18 in SEQ ID NO. 8 (such as with a more hydrophobic residue). In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 modified by substitutions in the amino acids corresponding to L8, V12, and F15 in SEQ ID NO. 8 (such as with more hydrophobic residues). In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID No. 4, modified by one or more substitutions corresponding to the following substitutions in SEQ ID No. 8: Y1Q, Y2L, Y2I, M3V, M3I, L5C, L8F, V12F, V13Y, F15S, a16V, a16I, I18V, I18L, C19M, C20M, and C21G. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 modified by substitutions corresponding to the Y2L, M3V, A16V and I18V substitutions in SEQ ID NO. 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 modified by substitutions corresponding to the Y2I, M3I, A16I and I18L substitutions in SEQ ID NO. 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of the transmembrane domain contained in SEQ ID NO. 4 modified by substitutions corresponding to the L8F, V12F, and F15S substitutions in SEQ ID NO. 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of any one of SEQ ID Nos. 14-26.

In some embodiments, a non-naturally occurring TCR-TM derived from a T cell receptor described herein comprises, consists essentially of, or consists of: the transmembrane domain of the gamma TCR subunit comprising the amino acid sequence of SEQ ID NO 8, which is modified by substitution of one or more amino acid residues. In some embodiments, the transmembrane domain of the gamma TCR subunit is modified by substitution of NO more than 5 amino acid residues in SEQ ID No. 8. In some embodiments, the transmembrane domain of the gamma TCR subunit is modified by substitution of a single amino acid residue in SEQ ID NO 8. In some embodiments, at least one of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, each of the substituted amino acids is substituted with a residue that is more hydrophobic than the corresponding unsubstituted residue. In some embodiments, at least one of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3. In some embodiments, each of the substituted amino acids is adjacent to an amino acid in the TCRM that is involved in binding CD 3.

In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO:8 modified by one or more substitutions (such as with a more hydrophobic residue) of amino acids corresponding to the following amino acids in SEQ ID NO: 8: y1, Y2, M3, L5, L8, V12, V13, F15, a16, I18, C19, C20 and C21. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO:8 modified by substitutions (such as with more hydrophobic residues) in the amino acids corresponding to Y2, M3, a16, and I18 in SEQ ID NO: 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO:8 modified by substitutions in the amino acids corresponding to L8, V12, and F15 in SEQ ID NO:8 (such as with more hydrophobic residues). In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID No. 8 modified by one or more substitutions corresponding to the following substitutions in SEQ ID No. 8: Y1Q, Y2L, Y2I, M3V, M3I, L5C, L8F, V12F, V13Y, F15S, a16V, a16I, I18V, I18L, C19M, C20M, and C21G. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO 8 modified by substitutions corresponding to the Y2L, M3V, A16V, and I18V substitutions in SEQ ID NO 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO 8 modified by substitutions corresponding to the Y2I, M3I, A16I, and I18L substitutions in SEQ ID NO 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of SEQ ID NO 8 modified by substitutions corresponding to the L8F, V12F, and F15S substitutions in SEQ ID NO 8. In some embodiments, the non-naturally occurring TCR-TM comprises the amino acid sequence of any one of SEQ ID NOs 14-26.

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising, consisting essentially of, or consisting of a first TCR-TM comprising any one of the amino acid sequences of SEQ ID NOs 7 and 9-13; and a second TCRD comprising, consisting essentially of, or consisting of a second TCR-TM comprising, consisting of the amino acid sequence of any one of SEQ ID NOs 8 and 14-26, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCR-TM and the second TCR-TM are selected according to any one of the caTCRs listed in Table 2. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linking peptide comprises the amino acid sequence of SEQ ID NO 31 or 32 or a variant thereof, and/or the second linking peptide comprises the amino acid sequence of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the second TCR intracellular domain comprises the amino acid sequence of SEQ ID NO 36 or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof.In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv).

TABLE 2

Antigen binding modules

In some embodiments, the antigen binding moiety is an antibody moiety according to any of the cadrs described herein. In some embodiments, the antibody moiety is selected from the group consisting of: fab, Fab ', (Fab')2, Fv and single chain Fv (scFv). In some embodiments, when the antibody moiety is a multimer comprising a first antibody moiety chain and a second antibody moiety chain, the caTCR comprises a first TCRD linked to the first or second antibody moiety chain and a second TCRD linked to the other antibody moiety chain. In some embodiments, the antibody moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the antibody moiety specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof.

In some embodiments, an antibody is directed against any of the caTCRs described hereinOriginal bonding module of_H1 and C_LAn antibody portion of the domain. In some embodiments, C_HThe 1 domain is derived from an IgG (e.g., IgG1, IgG2, IgG3, or IgG4) heavy chain, optionally human. In some embodiments, C _H1 domain is a variant that includes one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived. In some embodiments, C_HDomain 1 comprises the amino acid sequence of any one of SEQ ID NOs 37-47 or variants thereof. In some embodiments, C_HDomain 1 comprises the amino acid sequence of SEQ ID NO 37 or a variant thereof. In some embodiments, C_LThe domains are derived from a kappa or lambda light chain, optionally human. In some embodiments, C_LA domain is a variant that includes one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived. In some embodiments, C_LThe domain includes the amino acid sequence of SEQ ID NO 48 or a variant thereof. In some embodiments, C _H1 and/or C_LThe domains include one or more modifications that do not substantially alter their binding affinity for each other. In some embodiments, C _H1 and/or C_LThe domains include one or more modifications that increase their binding affinity to each other and/or introduce non-naturally occurring disulfide bonds.

In some embodiments, any of the caTCRs described herein comprises an antibody portion that specifically binds to a target antigen, the antibody portion comprising a CDR or variable domain (V) of the antibody portion specific for the target antigen_HAnd/or V_LA domain). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomains) (see, e.g., WO2017066136a 2). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 58; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 59Sequence composition; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD20_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 60; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 61; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (see, e.g., USSN 62/650,955 filed on 3/30/2018, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 101; and/or V _L102, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (see, e.g., USSN 62/490,586 filed on 26/4/2017, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (e.g. V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 64; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 65; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR1_HAnd/or V_LDomains) (see, e.g., WO2016/187220 and WO 2016/18721)6). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR2_HAnd/or V_LDomains) (see, e.g., WO 2016/142768). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for BCMA_HAnd/or V_LDomains) (see, e.g., WO2016/090327 and WO 2016/090320). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for GPRC5D_HAnd/or V_LDomains) (see, e.g., WO2016/090329 and WO 2016/090312). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for FCRL5_HAnd/or V_LDomains) (see, e.g., WO 2016/090337). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of an antibody moiety specific for WT-1_HAnd/or V_LDomains) (see, e.g., WO2012/135854, WO2015/070078, and WO 2015/070061). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of the antibody moiety specific for AFP_HAnd/or V_LDomains) (see, e.g., WO 2016/161390). In some embodiments, the antibody portion comprises CDRs or variable domains (V) of an antibody portion specific for HPV16-E7_HAnd/or V_LDomains) (see, e.g., WO 2016/182957). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for NY-ESO-1_HAnd/or V_LDomains) (see, e.g., WO 2016/210365). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PRAME_HAnd/or V_LDomains) (see, e.g., WO 2016/191246). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for EBV-LMP2A_HAnd/or V_LDomains) (see, e.g., WO 2016/201124). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for KRAS_HAnd/or V_LDomains) (see, e.g., WO 2016/154047). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PSA_HAnd/or V_LDomain) (see, e.g., WO2017/015634). In some embodiments, the antibody moiety is a Fab comprising: a Fab chain comprising V_HDomain and C _H1 domain; and another Fab chain comprising V_LDomain and C_LA domain. In some embodiments, C_HDomain 1 comprises the amino acid sequence of any one of SEQ ID NOS 37-47, and/or C_LThe domain includes the amino acid sequence of SEQ ID NO 48. In some embodiments, C _H1 domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 37 and C_LThe domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 48.

caTCR construct

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antibody moiety that specifically binds to the target antigen, wherein the antibody moiety is linked to the first and/or second TCRD. In some embodiments, the antibody moiety is selected from the group consisting of: fab, Fab ', (Fab')2, Fv and single chain Fv (scFv). In some embodiments, the antibody moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the antibody moiety specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the stabilizing moiety is located between the TCRM and the antibody moiety. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising, consisting essentially of, or consisting of a first TCR-TM comprising any one of the amino acid sequences of SEQ ID NOs 7 and 9-13; and a second TCRD comprising, consisting essentially of, or consisting of a second TCR-TM comprising, consisting of the amino acid sequence of any one of SEQ ID NOs 8 and 14-26, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antibody moiety that specifically binds to the target antigen, wherein the antibody moiety is linked to the first and/or second TCRD. In some embodiments, the first TCR-TM and the second TCR-TM are selected according to any one of the caTCRs listed in Table 2. In some embodiments, the antibody moiety is selected from the group consisting of: fab, Fab ', (Fab')2, Fv and single chain Fv (scFv). In some embodiments, the antibody moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the antibody moiety specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linking peptide comprises the amino acid sequence of SEQ ID NO 31 or 32 or a variant thereof, and/or the second linking peptide comprises the amino acid sequence of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the second TCR intracellular domain comprises the amino acid sequence of SEQ ID NO 36 or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, the caTCR comprising: a) a first TCRD comprising a first TCR-TM; and a second TCRD comprising a second TCR-TM, wherein the first and second TCR-TMs each comprise, consist essentially of, or consist of: 7 and 8, 9 and 8, 7 and 14, 7 and 15, 7 and 16, 10 and 16, 7 and 17, 7 and 18, 7 and 19, 7 and 20, 7 and 21, 7 and 22, 11 and 23, 12 and 24, 7 and 25, or 13 and 26, and wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antibody moiety that specifically binds to the target antigen, wherein the antibody moiety is linked to the first and/or second TCRD. For example, in some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first TCRD comprising, consisting essentially of, or consisting of a first TCR-TM comprising, consisting of the amino acid sequence of SEQ ID NO. 7; and a second TCRD comprising, consisting essentially of, or consisting of a second TCR-TM comprising, consisting of the amino acid sequence of SEQ ID NO. 8, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) an antibody moiety that specifically binds to the target antigen, wherein the antibody moiety is linked to the first and/or second TCRD. In some embodiments, the antibody moiety is selected from the group consisting of: fab, Fab ', (Fab')2, Fv and single chain Fv (scFv). In some embodiments, the antibody moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the antibody moiety specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linking peptide comprises the amino acid sequence of SEQ ID NO 31 or 32 or a variant thereof, and/or the second linking peptide comprises the amino acid sequence of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the second TCR intracellular domain comprises the amino acid sequence of SEQ ID NO 36 or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first polypeptide chain comprising a first antigen binding domain comprising a first Fab chain linked to a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a TCR; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a second Fab chain linked to a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule, and wherein the first and second Fab chains form a Fab-like antigen binding moiety that specifically binds a target antigen. In some embodiments, a) the first Fab chain comprises V_HAnd C _H1 antibody domain and the second Fab chain comprises V_LAnd C_LAn antibody domain; or b) the first Fab chain comprises V_LAnd C_LAn antibody domain and the second Fab chain comprises V_HAnd C _H1 antibody domain. For example, in some embodiments, the caTCR comprises: a) a first polypeptide chain comprising V linked to a first TCRD_HAnd C_H1 a first Fab chain of an antibody domain; and b) a second Fab chain linked to a second TCRD comprising V_LAnd C_LAn antibody domain. In some embodiments, the caTCR comprises: a) a first Fab chain linked to a first TCRD comprising V_LAnd C_LAn antibody domain; and b) a second Fab chain linked to a second TCRD comprising V_HAnd C _H1 antibody domain. In some embodiments, a peptide linker is present between one or both of the TCRDs and the Fab chain to which they are attached. In some embodiments, at C _H1 domain with C_LDisulfide bonds exist between residues in the domains. In some embodiments, C _H1 and/or C_LThe domain includes one or more modifications that increase the binding affinity of the Fab chains to each other. In some embodiments, exchange C _H1 and C_LDomain such that one of the Fab chains comprises V_HAnd C_LAntibody domain, and the other Fab chain comprises V_LAnd C _H1 antibody domain. In some embodiments, FabThe like antigen binding modules specifically bind to cell surface antigens, including but not limited to CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the Fab-like antigen binding module specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first or second stabilizing domain is located between the first TCRD and the Fab chain to which it is attached, and the other stabilizing domain is located between the second TCRD and the Fab chain to which it is attached. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first polypeptide chain comprising a first antigen binding domain comprising a first Fab chain linked to a first TCRD comprising a first TCR-TM comprising, consisting essentially of, or consisting of the amino acid sequences of any one of SEQ ID NOs 7 and 9-13; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a second Fab chain linked to a second TCRD comprising, consisting essentially of, or consisting of the amino acid sequence comprising any one of SEQ ID NOs 8 and 14-26, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule, and wherein the first and second Fab chains form a Fab-like antigen binding module that specifically binds to a target antigen. In some embodiments, the first TCR-TM and the second TCR-TM are selected according to any one of the caTCRs listed in Table 2. In some embodiments, a) the first Fab chain comprises V_HAnd C _H1 antibody domain and the second Fab chain comprises V_LAnd C_LAn antibody domain; or b) a first FabThe chain comprising V_LAnd C_LAn antibody domain and the second Fab chain comprises V_HAnd C _H1 antibody domain. In some embodiments, C_HDomain 1 comprises the amino acid sequence of any one of SEQ ID NOS 37-47, and/or C_LThe domain includes the amino acid sequence of SEQ ID NO 48. In some embodiments, C_H37, and C_L48, consists essentially of, or consists of the amino acid sequence of SEQ ID NO. In some embodiments, the Fab-like antigen binding moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the Fab-like antigen binding module specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linking peptide comprises the amino acid sequence of SEQ ID NO 31 or 32 or a variant thereof, and/or the second linking peptide comprises the amino acid sequence of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the second TCR intracellular domain comprises the amino acid sequence of SEQ ID NO 36 or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodimentsThe first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first polypeptide chain comprising a first antigen binding domain comprising a first Fab chain linked to a first TCRD comprising a first TCR-TM and a second polypeptide chain comprising a second antigen binding domain comprising a second Fab chain linked to a second TCRD comprising a second TCR-TM, wherein the first and second TCR-TM comprise, consist essentially of, or consist of, respectively: 7 and 8, 9 and 8, 7 and 14, 7 and 15, 7 and 16, 10 and 16, 7 and 17, 7 and 18, 7 and 19, 7 and 20, 7 and 21, 7 and 22, 11 and 23, 12 and 24, 7 and 25 or 13 and 26, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and wherein the first and second Fab chains form a Fab-like antigen binding module that specifically binds to a target antigen. In some embodiments, a) the first Fab chain comprises V_HAnd C _H1 antibody domain and the second Fab chain comprises V_LAnd C_LAn antibody domain; or b) the first Fab chain comprises V_LAnd C_LAn antibody domain and the second Fab chain comprises V_HAnd C _H1 antibody domain. In some embodiments, C_HDomain 1 comprises the amino acid sequence of any one of SEQ ID NOS 37-47, and/or C_LThe domain includes the amino acid sequence of SEQ ID NO 48. In some embodiments, C_H37, consists essentially of, or consists ofAnd C is_L48, consists essentially of, or consists of the amino acid sequence of SEQ ID NO. In some embodiments, the Fab-like antigen binding moiety specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the Fab-like antigen binding module specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linking peptide comprises the amino acid sequence of SEQ ID NO 31 or 32 or a variant thereof, and/or the second linking peptide comprises the amino acid sequence of SEQ ID NO 33 or 34 or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the second TCR intracellular domain comprises the amino acid sequence of SEQ ID NO. 36 or a variant thereof. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain having the sequences of SEQ ID NOs 7 and 8. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some casesIn embodiments, a spacer moiety is present between any two caTCR moieties or domains.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and wherein the first and/or second TCR-TM is non-naturally occurring; and b) a Fab 'which specifically binds to the target antigen, wherein the Fab' comprises a V-containing group_H、C _H1 and part of the hinge antibody domain, and a first Fab' chain comprising V_LAnd C_LA second Fab ' chain of the antibody domain, and wherein the first Fab ' chain is linked to the first or second TCRD and the second Fab ' chain is linked to the other TCRD. In some embodiments, a peptide linker is present between one or both of the TCRDs and the Fab' chain to which they are attached. In some embodiments, at C _H1 domain with C_LDisulfide bonds exist between residues in the domains. In some embodiments, C _H1 and/or C_LThe domain includes one or more modifications that increase the binding affinity of the Fab' chains to each other. In some embodiments, exchange C _H1 and C_LDomain such that the first Fab' chain comprises V_H、C_LAnd a partial hinge antibody domain, and the second Fab' chain comprises V_LAnd C _H1 domain. In some embodiments, Fab' specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, Fab' specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including (but not limited to) WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first or second stability region is located at the firstTCRD is between its linked Fab 'chains and another stable is between the second TCRD and its linked Fab' chains. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) (Fab ')2 which specifically binds to a target antigen, wherein (Fab')2 comprises: a first and a second (Fab')2 chain comprising V_H、C _H1 and a partial hinge antibody domain; and a third and fourth (Fab')2 chain comprising V_LAnd C_LAn antibody domain, and wherein the first (Fab ')2 chain is linked to the first or second TCRD, and the second (Fab')2 chain is linked to the other TCRD. In some embodiments, a peptide linker is present between one or both of the TCRDs and the (Fab')2 chain to which it is attached. In some embodiments, at C _H1 domain with C_LDisulfide bonds exist between residues in the domains. In some embodiments, C _H1 and/or C_LThe domains include one or more modifications that increase the binding affinity of the (Fab')2 chains to each other. In some embodiments, exchange C _H1 and C_LDomain such that the first and second (Fab')2 chains comprise V_H、C_LAnd a partial hinge antibody domain, and the third and fourth (Fab')2 chains comprise V_LAnd C _H1 domain. In some embodiments, (Fab')2 specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, (Fab')2 specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the cadCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domainA domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the caTCR. In some embodiments, the first or second stabilizing domain is located between the first TCRD and the (Fab ')2 chain to which it is attached, and the other stabilizing domain is located between the second TCRD and the (Fab')2 chain to which it is attached. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen, comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an Fv that specifically binds to a target antigen, wherein the Fv comprises a peptide comprising V_HFirst Fv chain of antibody domain and V-containing_LA second Fv chain of an antibody domain, and wherein the first Fv chain is linked to the first or second TCRD and the second Fv chain is linked to the other TCRD. In some embodiments, a peptide linker is present between one or both of the TCRDs and the Fv chain to which it is attached. In some embodiments, the Fv specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the Fv specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first or second stability domain is located between the Fv chain to which the first TCRD is connected and the other stability domain is located between the Fv chain to which the second TCRD is connected. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, the present inventionThe cadCR described herein specifically binds a target antigen, comprising: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) a first scFv that specifically binds to a target antigen, wherein the first scFv comprises V_HAnd V_LAn antibody domain, and wherein the first scFv is linked to the first or second TCRD. In some embodiments, the caTCR further comprises a second antigen binding moiety linked to the first scFv or to the TCRD not linked to the first scFv. In some embodiments, the second antigen binding moiety specifically binds to the target antigen. In some embodiments, the second antigen binding moiety specifically binds to an antigen that is different from the target antigen. In some embodiments, the second antigen binding moiety is a second scFv. In some embodiments, a peptide linker is present between the first scFv and the TCRD to which it is attached or between the second antigen binding moiety and the scFv or TCRD to which it is attached. In some embodiments, the scFv specifically binds to a cell surface antigen, including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the scFv specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein including, but not limited to, WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first or second stabilizing domain is located between the first scFv and the TCRD to which it is attached, and the other stabilizing domain is attached to the second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

In some embodiments, a caTCR described herein specifically binds a target antigen comprising a) a first TCRD comprising a sourceA first TCR-TM from one of the transmembrane domains of the TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) a first scFv that specifically binds to the target antigen and a second scFv, wherein the first and second scFv comprise V_HAnd V_LAn antibody domain, and wherein the first scFv is linked to the first or second TCRD and the second scFv is linked to the other TCRD. In some embodiments, the second TCRD specifically binds to the target antigen. In some embodiments, the second scFv comprises, consists essentially of, or consists of the amino acid sequence of the first scFv. In some embodiments, the second scFv specifically binds to an antigen different from the target antigen. In some embodiments, the first and/or second scFv uniquely binds to a cell surface antigen, including but not limited to CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants and mutants thereof. In some embodiments, the first and/or second scFv individually specifically bind to a peptide/MHC complex, wherein the peptide is derived from a protein, including but not limited to WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first or second stabilizing domain is located between the first scFv and the TCRD to which it is attached, and the other stabilizing domain is located between the second scFv and the TCRD to which it is attached. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.

Multispecific catrp

In some embodiments, the caTCR is a multispecific caTCR that specifically binds to two or more (e.g., 2, 3, 4, or more) different target antigens or epitopes. In some embodiments, the multispecific caTCR specifically binds to two or more (e.g., 2, 3, 4, or more) different target antigens. In some embodiments, the multispecific caTCR specifically binds to two or more (e.g., 2, 3, 4, or more) different epitopes on the same target antigen. In some embodiments, the multispecific caTCR includes an antigen binding moiety for each antigen or epitope. In some embodiments, the multispecific caTCR includes more than two antigen-binding moieties for at least one antigen or epitope. In some embodiments, the multispecific caTCR comprises a multispecific antigen-binding moiety comprising two or more (e.g., 2, 3, 4, or more) antigen-binding domains that each specifically bind to an antigen or epitope. In some embodiments, the multispecific caTCR is bispecific. In some embodiments, the multispecific caTCR is trispecific.

A multispecific molecule is a molecule having binding specificity for at least two different antigens or epitopes (e.g., a bispecific antibody has binding specificity for two antigens or epitopes). Multispecific catrs having more than two valencies and/or specificities are also contemplated. Bispecific antibodies have been described, for example, in Brinkmann U. and Kontermann R.E. (2017) MABS,9(2), 182-. Trispecific antibodies can be prepared, see Tutt et al J.Immunol.147:60 (1991). It will be appreciated that one skilled in the art can select appropriate characteristics of individual multispecific molecules known in the art to form a multispecific caTCR.

In some embodiments, the caTCR (also referred to herein as a "multispecific caTCR") comprises: a) a multispecific (e.g. bispecific) antigen-binding module comprising a first antigen-binding domain that specifically binds to a first target antigen and a second antigen-binding domain that specifically binds to a second target antigen; and b) TCRMs including a first TCRD (TCRD1) comprising a first TCR-TM and a second TCRD (TCRD2) comprising a second TCR-TM; wherein the TCRM contributes to recruitment of at least one TCR-associated signaling molecule. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first linker peptide comprises a TCR subunit from which the first TCR-TM is derivedAll or a portion of the linker peptide of the unit or a variant thereof, and/or the second linker peptide comprises all or a portion of the linker peptide of the TCR subunit from which the second TCR-TM is derived or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the first TCR-TM is derived, and/or the second TCR endodomain comprises a sequence of an endodomain from a TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise an antibody moiety, such as C _H1 and C_LAn antibody domain, or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a naturally occurring α β T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, both the first TCR-TM and the second TCR-TM are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCR-TM comprises at most 5 amino acid substitutions (e.g., single amino acid substitutions) as compared to the transmembrane domain from which the first TCR-TM is derived,and/or the second TCR-TM comprises up to 5 amino acid substitutions (e.g., single amino acid substitutions) as compared to the transmembrane domain from which the second TCR-TM is derived. In some embodiments, the substituted amino acid in the first TCR-TM is adjacent to the substituted amino acid in the second TCR-TM. In some embodiments, one or more substituted amino acids are adjacent to an amino acid in the first or second TCR-TM that is involved in binding to CD 3. In some embodiments, one or more (e.g., each) substituted amino acid is more hydrophobic than its corresponding unsubstituted amino acid. In some embodiments, the first TCR-TM comprises the amino acid sequence of any one of SEQ ID NOs 7 and 9-13, and wherein the second TCR-TM comprises the amino acid sequence of any one of SEQ ID NOs 8 and 14-26.

Exemplary structures of bispecific caTCRs are shown in FIGS. 13A-13E, where the target antigens are CD19 and CD22, but those of skill in the art will readily appreciate that the same structural format can be used to make bispecific caTCRs that target other target antigens or epitopes.

For example, the Dual Variable Domain (DVD) derived from DVD IgG (see DiGiamarino et al, mAbs 3(5):487-494) can be used as the bispecific antigen binding module in the caTCR (FIG. 13A). Various linkers for fusing the outer and inner variable domains have been developed and optimized for use in DVD-Ig, which can be adapted for constructing bispecific caTCRs with DVD modules. However, the variable domain stacking approach in DVD modules can affect the folding and target binding affinity of the inner variable domains. The linker between the two variable domains and the order of the two variable domains can affect the efficacy of the caTCR.

In some embodiments, the caTCR comprises: a) a multispecific (e.g., bispecific) antigen-binding module comprising an Fv that specifically binds to a first target antigen and a Fab that specifically binds to a second target antigen; and b) TCRMs including a first TCRD (TCRD1) comprising a first TCR-TM and a second TCRD (TCRD2) comprising a second TCR-TM; wherein the TCRM contributes to recruitment of at least one TCR-associated signaling molecule.

In some embodiments, the caTCR comprises: (i) a first polypeptide chain comprising N-terminus to C-terminus: v_H1-L1-V_H2-C_H1-TCRD1; and a second polypeptide chain comprising N-terminus to C-terminus: v_L1-L2-V_L2-C_L-TCRD 2; (ii) a first polypeptide chain comprising N-terminus to C-terminus: v_H1-L1-V_L2-C_L-TCRD 1; and a second polypeptide chain comprising N-terminus to C-terminus: v_L1-L2-V_H2-C_H1-TCRD 2; (iii) a first polypeptide chain comprising N-terminus to C-terminus: v_L1-L1-V_H2-C_H1-TCRD 1; and a second polypeptide chain comprising N-terminus to C-terminus: v_H1-L2-V_L2-C_L-TCRD 2; or (iv) a first polypeptide chain comprising N-terminus to C-terminus: v_L1-L1-V_L2-C_L-TCRD 1; and a second polypeptide chain comprising N-terminus to C-terminus: v_H1-L2-V_H2-C_H1-TCRD2 wherein V_H1 and V_L1 forms a first antigen binding domain that specifically binds to a first target antigen, and V_H2 and V_L2 forming a second antigen binding domain that specifically binds to a second target antigen, wherein TCRD1 and TCRD2 form a TCRM that contributes to recruitment of at least one TCR-associated signaling molecule, and wherein L1 and L2 are peptide linkers. In some embodiments, L1 and/or L2 is about 5 to about 50 (e.g., about 5-10, about 10-15, or about 15-30) amino acids in length. In some embodiments, L1 and L2 have the same length. In some embodiments, L1 and L2 have the same amino acid sequence. In some embodiments, L1 and L2 have different lengths. In some embodiments, L1 and L2 have different amino acid sequences. An exemplary bispecific caTCR is shown in fig. 13A.

Cross-double variable domains (CODV) derived from CODV-IgG (see Steinmetz et al; mAbs (2016),8(5):867-878) can be used as the bispecific antigen binding module in the caTCR (FIG. 13B). CODV allows each Fv to have a relatively unobstructed antigen binding site. Various linkers for fusing the heavy and light chain variable regions have been developed and optimized for use with CODV-Ig, which can be suitable for constructing bispecific caTCRs with a CODV module. However, proper folding of CODV modules can be challenging, and long linkers used in CODV modules can be a potential source of immunogenicity and are susceptible to protein cleavage.

In some embodiments, the caTCR comprises: a) multiple specificity (example)E.g., bispecific) an antigen-binding module comprising a first Fv that specifically binds to a first target antigen and a second Fv that specifically binds to a second target antigen; and b) TCRMs including a first TCRD (TCRD1) comprising a first TCR-TM, and a second TCRD (TCRD2) comprising a second TCR-TM; wherein the TCRM contributes to recruitment of at least one TCR-associated signaling molecule. In some embodiments, the caTCR further comprises C _H1 and C_L。

In some embodiments, the caTCR comprises: (i) a first polypeptide chain comprising N-terminus to C-terminus: v_H1-L1-V_H2-C_H1-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: v_L2-L2-V_L1-C_L-TCRD 2; or (ii) a first polypeptide chain comprising N-terminus to C-terminus: v_L1-L1-V_L2-C_L-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: v_H2-L2-V_H1-C_H1-TCRD2 wherein V _H1 and V _L1 forms a first antigen binding domain that specifically binds to a first target antigen, and V _H2 and V _L2 forming a second antigen binding domain that specifically binds to a second target antigen, wherein TCRD1 and TCRD2 form a TCRM that contributes to recruitment of at least one TCR-associated signaling molecule, and wherein L1 and L2 are peptide linkers. In some embodiments, L1 and/or L2 is about 5 to about 50 (e.g., about 5-20, about 15-30, or about 30-50) amino acids in length. In some embodiments, L1 and L2 have the same length. In some embodiments, L1 and L2 have the same amino acid sequence. In some embodiments, L1 and L2 have different lengths. In some embodiments, L1 and L2 have different amino acid sequences. An exemplary bispecific caTCR is shown in fig. 13B.

Bispecific antigen binding modules derived from scFv fusion proteins (such as those described in Chen et al, mAbs 8(4): 761-774) can be used in bispecific caTCRs (FIG. 13C). Expression of bispecific antibodies with a similar fusion format has demonstrated proper folding and stability of this format. Various linkers for fusing scFv to constant domains have been developed and optimized for use in these bispecific antibodies, which can be suitable for constructing bispecific catrs with similar scFv fusion domains. However, steric hindrance between scfvs may compromise binding of the scFv to its target antigen.

In some embodiments, the caTCR comprises: a) a multispecific (e.g., bispecific) antigen-binding moiety comprising a first scFv that specifically binds to a first target antigen and a second scFv that specifically binds to a second target antigen; and b) TCRMs including a first TCRD (TCRD1) comprising a first TCR-TM and a second TCRD (TCRD2) comprising a second TCR-TM; wherein the TCRM contributes to recruitment of at least one TCR-associated signaling molecule. In some embodiments, the caTCR further comprises C _H1 and C_L。

In some embodiments, the caTCR comprises: (i) a first polypeptide chain comprising N-terminus to C-terminus: scFv1-L1-C_H1-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: scFv2-L2-C_L-TCRD 2; or (ii) a first polypeptide chain comprising N-terminus to C-terminus: scFv2-L1-C_H1-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: scFv1-L2-C_L-TCRD2, wherein scFv1 specifically binds to a first target antigen and scFv2 specifically binds to a second target antigen, wherein TCRD1 and TCRD2 form a TCRM that contributes to recruitment of at least one TCR-associated signaling molecule, and L1 and L2 are peptide linkers. In some embodiments, L1 and/or L2 is about 5 to about 50 (e.g., about 5-10, about 10-15, or about 15-30) amino acids in length. In some embodiments, L1 and L2 have the same length. In some embodiments, L1 and L2 have the same amino acid sequence. In some embodiments, L1 and L2 have different lengths. In some embodiments, L1 and L2 have different amino acid sequences. An exemplary bispecific caTCR is shown in fig. 13C.

Bispecific antigen binding modules derived from IgG-scFv bispecific antibodies or Fab-scFv-Fc bispecific antibodies can be used in bispecific caTCRs. In one format (FIG. 13D), scFv was attached to V of Fab_HOr V_LIt allows greater flexibility of the scFv and thus greater proximity of the Fab to its target antigen. However, scFv-Fab modules can have stability issues. In the second format (fig. 13E), Fab was fused to the first TCRD and scFv was fused to the second TCRD.

In some embodiments, the caTCR comprises: a) a multispecific (e.g., bispecific) antigen-binding module comprising an scFv that specifically binds to a first target antigen and a Fab that specifically binds to a second target antigen; and b) TCRMs including a first TCRD (TCRD1) comprising a first TCR-TM and a second TCRD (TCRD2) comprising a second TCR-TM; wherein the TCRM contributes to recruitment of at least one TCR-associated signaling molecule.

In some embodiments, the caTCR comprises: (i) a first polypeptide chain comprising N-terminus to C-terminus: scFv-L1-V_H-C_H1-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: v_L-C_L-TCRD 2; or (ii) a first polypeptide chain comprising N-terminus to C-terminus: v_H-C_H1-TCRD1, and a second polypeptide chain comprising N-terminus to C-terminus: scFv-L2-V_L-C_L-TCRD 2; wherein the scFv specifically binds to a first target antigen, and V_HAnd V_LForming a second antigen binding domain that specifically binds to a second target antigen, wherein TCRD1 and TCRD2 form a TCRM that contributes to recruitment of at least one TCR-associated signaling molecule, and wherein L1 and L2 are peptide linkers. In some embodiments, L1 and/or L2 is about 5 to about 50 (e.g., about 5-10, about 10-15, or about 15-30) amino acids in length. An exemplary bispecific caTCR is shown in fig. 13D.

In some embodiments, the caTCR comprises: (i) a first polypeptide chain comprising N-terminus to C-terminus: v_L-C_L-L1-TCRD1, a second polypeptide chain comprising N-terminus to C-terminus: v_H-C_H1, and a third polypeptide chain comprising N-terminus to C-terminus: scFv-L2-TCRD 2; (ii) a first polypeptide chain comprising N-terminus to C-terminus: v_H-C_H1-L1-TCRD1, a second polypeptide chain comprising N-terminus to C-terminus: v_L-C_LAnd a third polypeptide chain comprising N-terminus to C-terminus: scFv-L2-TCRD 2; (iii) a first polypeptide chain comprising N-terminus to C-terminus: scFv-L1-TCRD1, a second polypeptide chain comprising N-terminus to C-terminus: v_H-C_H1, and a third polypeptide chain comprising N-terminus to C-terminus: v_L-C_L-L2-TCRD 2; or (iv) a first polypeptide chain comprising an N-terminus to a C-terminus: scFv-L1-TCRD1, a second polypeptide chain comprising N-terminus to C-terminus: v_L-C_LAnd a third polypeptide chain comprising N-terminus to C-terminus: v_H-C_H1-L2-TCRD2(ii) a Wherein the scFv specifically binds to a first target antigen, and V_HAnd V_LForming a second antigen binding domain that specifically binds to a second target antigen, wherein TCRD1 and TCRD2 form a TCRM that contributes to recruitment of at least one TCR-associated signaling molecule, and wherein L1 and L2 are peptide linkers. In some embodiments, L1 and/or L2 is about 5 to about 50 (e.g., about 5-10, about 10-15, or about 15-30) amino acids in length. An exemplary bispecific caTCR is shown in fig. 13E. The length of the peptide linker between scFv and TCRD and between Fab and TCRD can be optimized because it can affect the accessibility of scFv and Fab to their target antigens.

The multispecific antigen-binding moiety of the multispecific caTCR may specifically bind to any suitable combination of target antigens or epitopes. In some embodiments, the multispecific antigen-binding moiety specifically binds to at least one cell surface antigen. In some embodiments, the at least one cell surface antigen is selected from the group consisting of: CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL5, including variants or mutants thereof. In some embodiments, the multispecific antigen-binding moiety specifically binds to at least one peptide/MHC complex. In some embodiments, the at least one peptide/MHC complex comprises a peptide derived from a protein selected from the group consisting of: WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, HIV-1, KRAS, histone H3.3, and PSA, including variants or mutants thereof. In some embodiments, the multispecific antigen-binding moiety specifically binds to a first cell surface antigen and a second cell surface antigen. In some embodiments, the multispecific antigen-binding moiety specifically binds to CD19 and CD 22. In some embodiments, the multispecific antigen-binding moiety specifically binds to CD19 and CD 20. In some embodiments, the multispecific antigen-binding moiety specifically binds to a first peptide/MHC complex and a second peptide/MHC complex. In some embodiments, the multispecific antigen-binding module specifically binds to a cell surface antigen and a peptide/MHC complex.

Chimeric co-stimulatory receptor (CSR) constructs

The ligand-specific chimeric co-stimulatory receptors (CSRs) described herein specifically bind to a target ligand (such as a cell surface antigen or peptide/MHC complex) and are capable of stimulating immune cells on the surface that functionally express the CSR upon target ligand binding. CSR comprises a ligand binding module providing ligand binding specificity, a transmembrane module, and a co-stimulatory immune cell signaling module allowing the stimulation of immune cells. CSR lacks functional primary immune cell signaling sequences. In some embodiments, the CSR lacks any primary immune cell signaling sequences. In some embodiments, the CSR comprises a single polypeptide chain comprising a ligand binding module, a transmembrane module, and a costimulatory signaling module. In some embodiments, the CSR comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form a ligand binding module, a transmembrane module, and a costimulatory signaling module. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the CSR is a multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide bond or by another chemical bond (such as a disulfide bond). In some embodiments, the first polypeptide chain is linked to the second polypeptide chain by at least one disulfide bond. In some embodiments, expression of CSR in the caTCR plus CSR immune cells is inducible. In some embodiments, expression of CSR in the caTCR plus CSR immune cells is inducible following signaling via the caTCR.

Examples of co-stimulatory immune cell signaling domains for CSRs of the invention include cytoplasmic sequences of co-receptors for T Cell Receptors (TCRs) that can function in concert with the calcrs to trigger signal transduction following engagement of the calcr, as well as any derivative or variant of these sequences and any synthetic sequence with the same functional capability.

In some cases, the signal generated via the TCR alone is insufficient to fully activate the T cell and a secondary or co-stimulatory signal is also required. Thus, in some embodiments, T cell activation is mediated by two distinct classes of intracellular signaling sequences: those that elicit antigen-dependent primary activation via the TCR (referred to herein as "primary T cell signaling sequences"); and those that function in an antigen-independent manner to provide a secondary or costimulatory signal (referred to herein as "costimulatory T cell signaling sequences").

Primary immune cell signaling sequences that function in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of primary ITAM-containing immune cell signaling sequences include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, and CD66 d. A "functional" primary immune cell signaling sequence is a sequence capable of transducing an immune cell activation signal when operably coupled to an appropriate recipient. A "non-functional" primary immune cell signaling sequence, which may include a fragment or variant of the primary immune cell signaling sequence, is unable to transduce an immune cell activation signal. The CSRs described herein lack functional primary immune cell signaling sequences, such as functional signaling sequences including ITAMs. In some embodiments, the CSR lacks any primary immune cell signaling sequences.

The co-stimulatory immune cell signaling sequence may be part of the intracellular domain of a co-stimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

In some embodiments, the target ligand is a cell surface antigen. In some embodiments, the target ligand is a peptide/MHC complex. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a molecule presented on the surface of a cell presenting the target antigen. For example, in some embodiments, the target antigen of the caTCR is a cancer-associated antigen presented on a cancer cell, and the target ligand is a ubiquitous molecule, such as an integrin, expressed on the surface of the cancer cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2.

In some embodiments, the ligand binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antibody moiety specifically binds to a cell surface antigen including, but not limited to, CD19, CD20, CD22, CD47, GPC-3, ROR1, ROR2, BCMA, GPRC5D, and FCRL 5. In some embodiments, the antibody moiety specifically binds to a peptide/MHC complex, wherein the peptide is derived from a protein including, but not limited to, WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomains) (see, e.g., WO2017066136a 2). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 58; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 59; or the CDRs contained therein).In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD20_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 60; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 61; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (see, e.g., USSN 62/650,955 filed on 3/30/2018). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 101; and/or V _L102, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (see, e.g., USSN 62/490,586 applied for 26/4/2017). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 64; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 65; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR1_HAnd/or V_LDomains) (see, e.g., WO2016/187220 and WO 2016/187216). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR2_HAnd/or V_LDomains) (see, e.g., WO 2016/142768). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for BCMA_HAnd/or V_LDomains) (see, e.g., WO2016/090327 and WO 2016/090320). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for GPRC5D_HAnd/or V_LDomains) (see, e.g., WO2016/090329 and WO 2016/090312). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for FCRL5_HAnd/or V_LDomains) (see, e.g., WO 2016/090337). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of an antibody moiety specific for WT-1_HAnd/or V_LDomains) (see, e.g., WO2012/135854, WO2015/070078, and WO 2015/070061). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of the antibody moiety specific for AFP_HAnd/or V_LDomains) (see, e.g., WO 2016/161390). In some embodiments, the antibody portion comprises CDRs or variable domains (V) of an antibody portion specific for HPV16-E7_HAnd/or V_LDomains) (see, e.g., WO 2016/182957). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for NY-ESO-1_HAnd/or V_LDomains) (see, e.g., WO 2016/210365). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PRAME_HAnd/or V_LDomains) (see, e.g., WO 2016/191246). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for EBV-LMP2A_HAnd/or V_LDomains) (see, e.g., WO 2016/201124). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for KRAS_HAnd/or V_LDomains) (see, e.g., WO 2016/154047). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PSA_HAnd/or V_LDomains) (see, e.g., WO 2017/015634).

In some embodiments, the ligand binding module is all or a portion of (or derived from) an extracellular domain of a receptor for a target ligand. In some embodiments, receptors include, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, and TIM-3.

In some embodiments, the transmembrane module comprises one or more transmembrane domains derived from, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154.

In some embodiments, the co-stimulatory signaling module comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell co-stimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the costimulatory signaling molecule includes a fragment of CD28, which includes the amino acid sequence of SEQ ID NO: 51. In some embodiments, the costimulatory signaling molecule includes a fragment of CD28, which includes the amino acid sequence of SEQ ID No. 52. In some embodiments, the costimulatory signaling molecule comprises a fragment of 4-1BB, which comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the costimulatory signaling molecule comprises a fragment of 4-1BB, which comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the costimulatory signaling molecule includes a fragment of CD8, which includes the amino acid sequence of SEQ ID No. 57. In some embodiments, the costimulatory signaling molecule includes a fragment of OX40, which includes the amino acid sequence of SEQ ID NO: 55. In some embodiments, the costimulatory signaling molecule includes a fragment of OX40 that includes the amino acid sequence of SEQ ID NO: 56. In some embodiments, the costimulatory signaling molecule includes a fragment of CD27, which includes the amino acid sequence of SEQ ID No. 86 or 87. In some embodiments, the costimulatory signaling molecule includes a fragment of CD30, which includes the amino acid sequence of SEQ id nos 88 or 89.

In some embodiments, the CSR further comprises a spacer module between any of the ligand binding module, transmembrane module, and costimulatory signaling module. In some embodiments, the spacer module comprises one or more peptide linkers connecting two CSR modules. In some embodiments, the spacer moiety comprises one or more peptide linkers between about 5 to about 70 amino acids in length (such as any of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any range between these values).

In some embodiments, the ligand binding moiety (such as an antibody moiety) specifically binds to the target antigen by: a) an affinity that is at least about 10 times (including, e.g., any of at least about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000, or more times) its binding affinity for other molecules; or b) no more than K for binding to other molecules_dA K of about 1/10 (such as no more than any one of about 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000, or less) of_d. Binding affinity can be determined by methods known in the art, such as ELISA, Fluorescence Activated Cell Sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). K_dCan be determined by methods known in the art, such as Surface Plasmon Resonance (SPR) assays using, for example, a Biacore instrument, or kinetic exclusion analysis using, for example, a Sapidyne instrument (KinExA).

In some embodiments, the CSRs described herein specifically bind to a target ligand (such as a cell surface antigen or peptide/MHC complex) comprising: a) a target Ligand Binding Domain (LBD); b) a transmembrane domain; and c) a co-stimulatory signaling domain, wherein the CSR is capable of stimulating immune cells on the surface that functionally express the CSR upon target ligand binding. In some embodiments, the target ligand is a cell surface antigen. In some embodiments, the target ligand is a peptide/MHC complex. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2. In some embodiments, the ligand binding domain is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the ligand binding domain is all or a portion of (or derived from) an extracellular domain of a receptor for a target ligand. In some embodiments, receptors include, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, and TIM-3. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a transmembrane protein comprising, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (frmp), wherein frmp comprises a CSR transmembrane domain. In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR comprises a fragment of an immune cell co-stimulatory molecule (fCSM), wherein the fCSM comprises a CSR transmembrane domain and a CSR co-stimulatory signaling domain. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains.

In some embodiments, a CSR described herein specifically binds to a target ligand, comprising: a) a target ligand binding domain; b) a transmembrane domain; and c) a co-stimulatory signaling domain, wherein the target ligand is a cell surface antigen, and wherein the CSR is capable of stimulating an immune cell on the surface that functionally expresses the CSR upon binding of the target ligand. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2. In some embodiments, the ligand binding domain is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the ligand binding domain is (or is derived from) all or a portion of an extracellular domain of a receptor for a target ligand. In some embodiments, receptors include, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, and TIM-3. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a transmembrane protein comprising, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (frmp), wherein frmp comprises a CSR transmembrane domain. In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR comprises a fragment of an immune cell co-stimulatory molecule (fCSM), wherein the fCSM comprises a CSR transmembrane domain and a CSR co-stimulatory signaling domain. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains.

In some embodiments, a CSR described herein specifically binds to a target ligand, comprising: a) a target ligand binding domain; b) a transmembrane domain; and c) a co-stimulatory signaling domain, wherein the target ligand is a peptide/MHC complex, and wherein the CSR is capable of stimulating an immune cell on the surface that functionally expresses the CSR upon binding of the target ligand. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the ligand binding domain is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a transmembrane protein comprising, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (frmp), wherein frmp comprises a CSR transmembrane domain. In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR comprises a fragment of an immune cell co-stimulatory molecule (fCSM), wherein the fCSM comprises a CSR transmembrane domain and a CSR co-stimulatory signaling domain. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains.

In some embodiments, a CSR described herein specifically binds to a target ligand (such as a cell surface antigen or peptide/MHC complex) comprising: a) a target ligand binding domain; b) a transmembrane domain; and c) a co-stimulatory signaling domain, wherein the ligand binding domain is an antibody moiety, and wherein the CSR is capable of stimulating an immune cell on the surface that functionally expresses the CSR upon target ligand binding. In some embodiments, the target ligand is a cell surface antigen. In some embodiments, the target ligand is a peptide/MHC complex. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a transmembrane protein comprising, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (frmp), wherein frmp comprises a CSR transmembrane domain. In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR comprises a fragment of an immune cell co-stimulatory molecule (fCSM), wherein the fCSM comprises a CSR transmembrane domain and a CSR co-stimulatory signaling domain. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains. In some embodiments, the CSR domain is selected according to any one of the CSRs listed in table 3.

TABLE 3

In some embodiments, the CSR comprises fCSM for CD 28. In some embodiments, the CSR comprises: a) a target ligand binding domain; b) a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 57; and c) a fragment of CD28 comprising the amino acid sequence of SEQ ID NO 52. In some embodiments, the CSR comprises: a) a target ligand binding domain; and b) a fragment of CD28 comprising the amino acid sequence of SEQ ID NO 51. In some embodiments, the CSR comprises: a target ligand binding domain and a CSR domain comprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the CSR comprises a 4-1BB fCSM. In some embodiments, the CSR comprises: a) a target ligand binding domain; b) a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 57; and c) a fragment of 4-1BB comprising the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CSR comprises: a) a target ligand binding domain; and b) a fragment of 4-1BB comprising the amino acid sequence of SEQ ID NO 53. In some embodiments, the CSR comprises: a target ligand binding domain and a CSR domain comprising the amino acid sequence of SEQ ID NO 91 or 92.

In some embodiments, the CSR comprises fCSM for CD 27. In some embodiments, the CSR comprises: a) a target ligand binding domain; b) a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 57; and c) a fragment of CD27 comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, the CSR comprises: a) a target ligand binding domain; and b) a fragment of CD27 comprising the amino acid sequence of SEQ ID NO 86. In some embodiments, the CSR comprises: a target ligand binding domain and a CSR domain comprising the amino acid sequence of SEQ ID NO 93 or 94.

In some embodiments, the CSR comprises fCSM for CD 30. In some embodiments, the CSR comprises: a) a target ligand binding domain; b) a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 57; and c) a fragment of CD30 comprising the amino acid sequence of SEQ ID NO. 89. In some embodiments, the CSR comprises: a) a target ligand binding domain; and b) a fragment of CD30 comprising the amino acid sequence of SEQ ID NO. 88. In some embodiments, the CSR comprises: a target ligand binding domain and a CSR domain comprising the amino acid sequence of SEQ ID NO 95 or 96.

In some embodiments, the CSR comprises fCSM of OX 40. In some embodiments, the CSR comprises: a) a target ligand binding domain; b) a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 57; and c) a fragment of OX40 comprising the amino acid sequence of SEQ ID NO 56. In some embodiments, the CSR comprises: a) a target ligand binding domain; and b) a fragment of OX40 comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the CSR comprises: a target ligand binding domain and a CSR domain comprising the amino acid sequence of SEQ ID NO 97 or 98.

In some embodiments, the CSRs described herein specifically bind to a target ligand (such as a cell surface antigen or peptide/MHC complex) comprising: a) a target ligand binding domain; b) a transmembrane domain; and c) a co-stimulatory signaling domain, wherein the ligand binding domain is (or is derived from) all or a portion of an extracellular domain of a receptor for the target ligand, and wherein the CSR is capable of stimulating immune cells on the surface that functionally express the CSR upon target ligand binding. In some embodiments, the target ligand is a cell surface antigen. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2. In some embodiments, the target ligand receptors comprise, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, and TIM-3. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, the costimulatory signaling domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; and b) a fragment of CD28 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 51 or 52. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 77. In some embodiments, the fragment of CD28 comprises the amino acid sequence of SEQ ID NO 51. In some embodiments, the CSR comprises the amino acid sequence of SEQ ID NO 90. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:103, and a fragment of CD 28. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 80.

In some embodiments, a CSR described herein specifically binds to CD20, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO 60_HDomain and V having amino acid sequence of SEQ ID NO 61_LA domain; and b) a fragment of CD28 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 51 or 52. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO. 78And (3) sequence composition. In some embodiments, the fragment of CD28 comprises the amino acid sequence of SEQ ID NO 51. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:103, and a fragment of CD 28. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 81.

In some embodiments, a CSR described herein specifically binds to GPC3, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO 64_HDomain and V having the amino acid sequence of SEQ ID NO 65_LA domain; and b) a fragment of CD28 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 51 or 52. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 79. In some embodiments, the fragment of CD28 comprises the amino acid sequence of SEQ ID NO 51. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:103, and a fragment of CD 28. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 82.

In some embodiments, a CSR described herein specifically binds to CD20, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO 60_HDomain and V having amino acid sequence of SEQ ID NO 61_LA domain; and b) a fragment of CD28 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 51 or 52. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:78Amino acid sequence composition. In some embodiments, the fragment of CD28 comprises the amino acid sequence of SEQ ID NO 51. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:103, and a fragment of CD 28. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 81.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; and b) a fragment of 4-1BB comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 53 or 54. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the fragment of 4-1BB comprises the amino acid sequence of SEQ ID NO 53. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO 104, and a fragment of 4-1 BB.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; b) a fragment of CD8 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID No. 57; and c) a fragment of 4-1BB that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO. 54. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, a fragment of CD8, and a fragment of 4-1 BB.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a)scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; b) a fragment of CD8 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID No. 57; and c) a fragment of OX40 that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO. 57. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, a fragment of CD8, and a fragment of OX 40.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; and b) a fragment of OX40 that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 56 or 57. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the fragment of OX40 comprises the amino acid sequence of SEQ ID NO 56. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, and a fragment of OX 40.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; b) a fragment of CD8 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID No. 57; and c) a fragment of CD27 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 87. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain comprising SEQ IPeptide linker of amino acid sequence of D NO 76 and V_HA domain. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, a fragment of CD8, and a fragment of CD 27.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; and b) a fragment of CD27 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 86 or 87. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the fragment of CD27 comprises the amino acid sequence of SEQ ID NO 86. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, and a fragment of CD 27.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; b) a fragment of CD8 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID No. 57; and c) a fragment of CD30 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO. 89. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, a fragment of CD8, and a fragment of CD 30.

In some embodiments, a CSR described herein specifically binds to CD19, the CSR comprising: a) scFv comprising V having the amino acid sequence of SEQ ID NO:58_HDomain and V having the amino acid sequence of SEQ ID NO 59_LA domain; and b) a fragment of CD30 comprising the amino acid sequence of SEQ ID NO:88 or 89, consists essentially of, or consists of the amino acid sequence. In some embodiments, the scFv comprises V from amino-terminus to carboxy-terminus_LDomain, peptide linker comprising the amino acid sequence of SEQ ID NO 76 and V_HA domain. In some embodiments, the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the CSR comprises, from amino-terminus to carboxy-terminus, an scFv, a peptide linker comprising SEQ ID NO:104, and a fragment of CD 30.

In some embodiments, expression of CSR in the caTCR plus CSR immune cells is inducible. In some embodiments, the caTCR plus CSR immune cell comprises a nucleic acid sequence encoding a CSR operably linked to an inducible promoter, comprising any of the inducible promoters described herein. In some embodiments, expression of CSR in the caTCR plus CSR immune cells is inducible following signaling via the caTCR. In some such embodiments, the caTCR plus CSR immune cells comprise a nucleic acid sequence encoding a CSR operably linked to a promoter or regulatory element in response to signaling via the caTCR. In some embodiments, the nucleic acid sequence encoding CSR is operably linked to a nuclear factor of an activated T cell (NFAT) -derived promoter. In some embodiments, the NFAT-derived promoter is the NFAT-derived minimal promoter (see, e.g., Durand, D et al, Molecular. cell. biol.8,1715-1724 (1988); Clipstone, NA, Crabtree, GR. Nature.1992357(6380): 695-7; Chmielewski, M. et al, Cancer research71.17(2011): 5697-. In some embodiments, the NFAT-derived promoter comprises the nucleotide sequence of SEQ ID NO 85. In some embodiments, the nucleic acid sequence encoding the CSR is operably linked to an IL-2 promoter.

Secretory Secondary Effector (SSE) constructs

In some embodiments, the caTCR plus CSR immune cells (such as T cells) are capable of secreting Secretory Secondary Effectors (SSE). Such immune cells are referred to herein as "caTCR plus CSR and SSE immune cells". The SSE enhances the immune response mediated by the caTCR plus CSR and SSE immune cells in which and from which the SSE is functionally expressed. In some embodiments, the SSE is capable of redirecting other immune cells (such as bystander T cells or NK cells) to target disease cells (such as target cancer cells). In some embodiments, the SSE is a multispecific antibody (such as a bispecific antibody) that targets immune cells (such as T cells or NK cells) and disease cells (such as cancer cells). In some embodiments, the SSE protects the caTCR plus CSR and SSE immune cells from an immunosuppressive environment (such as an immunosuppressive tumor environment). In some embodiments, the SSE provides autocrine activation of the caTCR plus CSR and stimulatory receptors on the SSE immune cells. In some embodiments, the SSE is an exogenous growth factor or a stimulatory cytokine. In some embodiments, expression of SSE in the caTCR plus CSR and SSE immune cells is inducible. In some embodiments, expression of SSE in the caTCR plus CSR and SSE immune cells is inducible following signaling via the caTCR.

In some embodiments, the SSE is a multispecific antibody (such as a bispecific antibody) that targets T cells and disease cells. In some embodiments, the SSE comprises an antibody moiety that specifically binds to a surface antigen of a T cell. In some embodiments, the T cell surface antigen is CD. In some embodiments, the SSE comprises an antibody moiety that specifically binds to a disease-associated antigen (such as a cancer-associated antigen). In some embodiments, the disease-associated antigen is a surface antigen of a disease cell (such as a cancer cell). In some embodiments, the disease-associated antigen is phosphoinositide-3 (GPC3), CD47, mucin-16 (MUC16), CD19, CD20, CD22, EpCAM, EGFR, HER2, CEA, PSMA, AFP, PSA, BCMA, FCRL5, NY-ESO, HPV16, or FoxP3, including variants or mutants thereof. In some embodiments, the SSE is a multispecific antibody selected from the group consisting of: tandem scFv, bifunctional antibodies (Db), single chain bifunctional antibodies (scDb), parental and force retargeting (DART) antibodies, and Double Variable Domain (DVD) antibodies. In some embodiments, the SSE is a bispecific antibody. In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting a T cell surface antigen and a second scFv targeting a disease-associated antigen.

In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD3 and a second scFv targeting a disease-associated antigen. In some embodiments, the disease-associated antigen is GPC3, CD47, MUC16, CD19, CD20, CD22, EpCAM, EGFR, HER2, CEA, PSMA, AFP, PSA, BCMA, FCRL5, NY-ESO, HPV16, or FoxP3, including variants or mutants thereof.

In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD3 and a second scFv targeting GPC 3. In some embodiments, the second scFv comprises: v_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 64; and V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 65. In some embodiments, V_HThe domain is at V_LThe amino terminus of the domain. In some embodiments, V_LThe domain is at V_HThe amino terminus of the domain. In some embodiments, the second scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 79. In some embodiments, the first scFv is amino-terminal to the second scFv. In some embodiments, the second scFv is amino-terminal to the first scFv. In some embodiments, the SSE comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 105.

In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD3 and a second scFv targeting CD 47. In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD3 and a second scFv targeting MUC 16.

In some embodiments, the SSE is a multispecific antibody (such as a bispecific antibody) that targets NK cells and disease-associated antigens (such as cancer-associated antigens). In some embodiments, the SSE comprises an antibody moiety that specifically binds to a surface antigen of an NK cell. In some embodiments, the NK cell surface antigen is CD16 a. In some embodiments, the SSE comprises an antibody moiety that specifically binds to a disease-associated antigen (such as a cancer-associated antigen). In some embodiments, the disease-associated antigen is a surface antigen of a disease cell (such as a cancer cell). In some embodiments, the disease-associated antigen is GPC3, CD47, MUC16, CD19, CD20, CD22, EpCAM, EGFR, HER2, CEA, PSMA, AFP, PSA, BCMA, FCRL5, NY-ESO, HPV16, or FoxP3, including variants or mutants thereof. In some embodiments, the SSE is a multispecific antibody selected from the group consisting of: tandem scFv, bifunctional antibodies (Db), single chain bifunctional antibodies (scDb), parental and force retargeting (DART) antibodies, and Double Variable Domain (DVD) antibodies. In some embodiments, the SSE is a bispecific antibody. In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting an NK cell surface antigen and a second scFv targeting a disease-associated antigen.

In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD16a and a second scFv targeting a disease-associated antigen. In some embodiments, the disease-associated antigen is GPC3, CD47, MUC16, CD19, CD20, CD22, EpCAM, EGFR, HER2, CEA, PSMA, AFP, PSA, BCMA, FCRL5, NY-ESO, HPV16, or FoxP3, including variants or mutants thereof.

In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD16a and a second scFv targeting GPC 3. In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD16a and a second scFv targeting CD 47. In some embodiments, the SSE is a tandem scFv comprising a first scFv targeting CD16a and a second scFv targeting MUC 16.

In some embodiments, an SSE described herein comprises an antibody portion that specifically binds to a disease-associated antigen, wherein the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for the disease-associated antigen_HAnd/or V_LA domain). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomains) (see, e.g., WO2017066136a 2). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD19_HAnd/or V_LDomain) (e.g., V)_HDomain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 58Consist of or consist of the amino acid sequence; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 59; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD20_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 60; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 61; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (see, e.g., USSN 62/650,955 filed on 3/30/2018, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for CD22_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 101; and/or V _L102, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO; or the CDRs contained therein). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (see, e.g., USSN 62/490,586 filed on 26/4/2017, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for GPC3_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 64; and/or V_LA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 65; or the CDRs contained therein). In thatIn some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR1_HAnd/or V_LDomains) (see, e.g., WO2016/187220 and WO 2016/187216). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for ROR2_HAnd/or V_LDomains) (see, e.g., WO 2016/142768). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for BCMA_HAnd/or V_LDomains) (see, e.g., WO2016/090327 and WO 2016/090320). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for GPRC5D_HAnd/or V_LDomains) (see, e.g., WO2016/090329 and WO 2016/090312). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for FCRL5_HAnd/or V_LDomains) (see, e.g., WO 2016/090337). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of an antibody moiety specific for WT-1_HAnd/or V_LDomains) (see, e.g., WO2012/135854, WO2015/070078, and WO 2015/070061). In some embodiments, the antibody moiety comprises a CDR or variable domain (V) of the antibody moiety specific for AFP_HAnd/or V_LDomains) (see, e.g., WO 2016/161390). In some embodiments, the antibody portion comprises CDRs or variable domains (V) of an antibody portion specific for HPV16-E7_HAnd/or V_LDomains) (see, e.g., WO 2016/182957). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antibody portion specific for NY-ESO-1_HAnd/or V_LDomains) (see, e.g., WO 2016/210365). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PRAME_HAnd/or V_LDomains) (see, e.g., WO 2016/191246). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for EBV-LMP2A_HAnd/or V_LDomains) (see, e.g., WO 2016/201124). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for KRAS_HAnd/or V_LDomain) (seeFor example WO 2016/154047). In some embodiments, the antibody portion comprises a CDR or variable domain (V) of the antibody portion specific for PSA_HAnd/or V_LDomains) (see, e.g., WO 2017/015634). In some embodiments, the antibody moiety is an scFv. In some embodiments, the SSE is a tandem scFv comprising: a) a first scFv that specifically binds to a surface antigen of a T cell (such as CD3) or NK cell (such as CD16 a); and b) an antibody moiety, wherein the antibody moiety is a second scFv. In some embodiments, the SSE comprises a first and a second scFv linked by a peptide linker. In some embodiments, the first scFv is amino-terminal to the second scFv. In some embodiments, the second scFv is amino-terminal to the first scFv.

In some embodiments, the SSE is a multispecific antibody (such as a bispecific antibody) that targets one or more soluble immunosuppressive agents. Such SSE may act as a capture agent to sequester soluble immunosuppressive agents from their targets, thereby reducing their immunosuppressive effects. In some embodiments, the SSE comprises one or more antibody moieties that specifically bind to one or more soluble immunosuppressive agents. In some embodiments, the immunosuppressive agent is an immunosuppressive cytokine. In some embodiments, immunosuppressive cytokines include TGF-beta family members (such as TGF-beta 1 to 4), IL-4, and IL-10, including variants or mutants thereof. In some embodiments, the SSE is a multispecific antibody selected from the group consisting of: tandem scFv, bifunctional antibodies (Db), single chain bifunctional antibodies (scDb), parental and force retargeting (DART) antibodies, and Double Variable Domain (DVD) antibodies. In some embodiments, the SSE is a bispecific antibody. For example, in some embodiments, the SSE is a tandem scFv comprising a first scFv targeting a first immunosuppressive cytokine (such as TGF β) and a second scFv targeting a second immunosuppressive cytokine (such as IL-4).

In some embodiments, the SSE is an antibody moiety that targets an immune checkpoint molecule. In some embodiments, the SSE is an antagonist of an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, CTLA-4, HVEM, BTLA, KIR, LAG-3, TIM-3 and A2 aR. In some embodiments, the SSE is an agonist of a stimulatory immune checkpoint molecule. In some embodiments, the stimulatory immune checkpoint molecule is selected from the group consisting of: CD28, ICOS, 4-1BB, OX40, CD27, and CD 40. In some embodiments, the antibody portion is a full-length antibody, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antibody moiety is an scFv.

In some embodiments, the SSE is an antagonistic antibody moiety targeting PD-1. In some embodiments, the antibody portion comprises a CDR or variable domain (V) of an antagonistic antibody portion specific for PD-1_HAnd/or V_LDomains) (see, e.g., WO 2016/210129). In some embodiments, the antagonistic antibody moiety is a full length antibody, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antagonistic antibody moiety is an scFv.

In some embodiments, the SSE is an antagonistic antibody moiety targeting CD 47. In some embodiments, the antagonistic antibody moiety comprises a CDR or variable domain (V) of the antagonistic antibody moiety specific for CD47_HAnd/or V_LDomain) (e.g., V)_HA domain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 66; and/or V_L67, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO; or the CDRs contained therein). In some embodiments, the antagonistic antibody moiety is a full length antibody, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antagonistic antibody moiety is an scFv.

In some embodiments, the SSE comprises an antibody moiety that binds to a target antigen: a) an affinity that is at least about 10 times (including, e.g., at least any of about 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000, or more times) its binding affinity for other molecules; or b) no more than K for binding to other molecules_dAbout 1/10 (such as no more than any of about 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000, or less)) K of_d. Binding affinity can be determined by methods known in the art, such as ELISA, Fluorescence Activated Cell Sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). K_dCan be determined by methods known in the art, such as Surface Plasmon Resonance (SPR) analysis using, for example, a Biacore instrument, or kinetic exclusion analysis using, for example, a Sapidyne instrument (KinExA).

In some embodiments, the SSE is a soluble molecule that specifically binds to a ligand of an immunosuppressive receptor. In some embodiments, the SSE comprises a ligand binding domain derived from the extracellular domain of an immunosuppressive receptor. In some embodiments, the ligand binding domain is part of an extracellular domain of a receptor. In some embodiments, the immunosuppressive receptor is selected from the group consisting of: FasR, TNFR1, TNFR2, SIRP alpha, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, CD40, and TIM-3.

In some embodiments, the SSE is a soluble molecule that specifically binds to and antagonizes an immunosuppressive receptor. In some embodiments, the SSE comprises a receptor binding domain derived from the extracellular domain of a ligand of an immunosuppressive receptor. In some embodiments, the receptor binding domain is part of the extracellular domain of the ligand. In some embodiments, the ligand is selected from the group consisting of: FasL, PD-L1, PD-L2, CD47, CD80, CD86, ICOSL, HVEM, 4-1BBL, OX40L, CD70, CD40L, and GAL 9.

In some embodiments, the SSE is an exogenous stimulatory cytokine. Exogenous cytokines described herein are cytokines expressed from exogenous genes. In some embodiments, exogenous stimulating cytokines are IL-12 family members. In some embodiments, the IL-12 family member is IL-12, IL-23, IL-27 or IL-35. In some embodiments, the exogenous stimulatory cytokine is IL-2, IL-15, IL-18, or IL-21. In some embodiments, the exogenous stimulus cytokine is capable of providing autocrine activation of receptors for the cytokine on the caTCR plus CSR and SSE immune cells.

In some embodiments, expression of SSE in the caTCR plus CSR and SSE immune cells is inducible. In some embodiments, the caTCR plus CSR and SSE immune cells comprise a nucleic acid sequence encoding an SSE operably linked to an inducible promoter, comprising any of the inducible promoters described herein. In some embodiments, expression of SSE in the caTCR plus CSR and SSE immune cells is inducible following signaling via the caTCR. In some such embodiments, the caTCR plus CSR and SSE immune cells comprise a nucleic acid sequence encoding an SSE operably linked to a promoter or regulatory element in response to signaling through the caTCR. In some embodiments, the nucleic acid sequence encoding the SSE is operably linked to a nuclear factor of an activated T cell (NFAT) -derived promoter. In some embodiments, the NFAT-derived promoter is the NFAT-derived minimal promoter (see, e.g., Durand, D et al, Molecular. cell. biol.8,1715-1724 (1988); Clipstone, NA, Crabtree, GR. Nature.1992357(6380): 695-7; Chmielewski, M. et al, cancer research71.17(2011): 5697-. In some embodiments, the NFAT-derived promoter comprises the nucleotide sequence of SEQ ID NO 85. In some embodiments, the nucleic acid sequence encoding the SSE is operably linked to the IL-2 promoter.

Nucleic acids

Also encompassed are nucleic acid molecules encoding the catrs, CSRs, and/or SSEs described herein. In some embodiments, a nucleic acid (set of nucleic acids) encoding a caTCR, CSR and/or SSE is provided according to any of the caTCR, CSR and SSE described herein.

The present invention also provides a vector into which the nucleic acid of the present invention is inserted.

Briefly, expression of the caTCR and/or CSR and/or SSE described herein by a nucleic acid encoding the caTCR and/or CSR and/or SSE can be achieved by: the nucleic acid is inserted into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR). The vector may be adapted for replication and integration in a eukaryotic host cell. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters suitable for regulating the expression of the desired nucleic acid sequence.

The nucleic acids of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. patent nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the invention provides gene therapy vectors.

Nucleic acids can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In addition, the expression vector may be provided to the 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 (2001, Molecular Cloning: A Laboratory Manual, Cold spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses suitable for use as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication, a promoter sequence, a suitable restriction endonuclease site, and one or more selectable markers that function in at least one organism (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Various virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. The selected gene can be inserted into a vector and encapsulated in a retroviral particle using techniques known in the art. Recombinant viruses can then be isolated and delivered to cells of an individual in vivo or ex vivo. Various retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Various adenoviral vectors are known in the art. In some embodiments, a lentiviral vector is used. Retroviral (such as lentiviral) derived vectors are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its spread in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from oncogenic retroviruses (such as murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. It also has the additional advantage of low immunogenicity.

Additional promoter elements (e.g., enhancers) regulate the transcription initiation frequency. Typically, these elements are located in the region 30-110bp upstream of the start site, but multiple promoters have recently been shown to contain functional elements also located downstream of the start site. The spacing between promoter elements is typically flexible such that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements may increase to 50bp apart before activity begins to decline.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high expression levels of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, monkey virus 40(SV40) early promoter, Mouse Mammary Tumor Virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, epstein barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of its operably linked polynucleotide sequence when such expression is desired or turning off expression when expression is not desired. Exemplary inducible promoter systems for use in eukaryotic cells include, but are not limited to, hormone regulatory elements (see, e.g., Mader, S. and White, J.H. (1993) Proc. Natl. Acad. Sci. USA90: 5603-; datta, R. et al (1992) Proc. Natl. Acad. Sci. USA89: 1014-. Other exemplary inducible promoter systems for use in mammalian systems in vitro or in vivo are described in Gingrich et al (1998) Annual Rev. Neurosci 21: 377-405.

An exemplary inducible promoter system for use in the present invention is the Tet system. Such systems are based on the Tet system described by Gossen et al (1993). In an exemplary embodiment, the polynucleotide of interest is under the control of a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) will bind to the TetO site and repress transcription from the promoter. In the active state, for example in the presence of an inducing agent such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or an active analog thereof, the inducing agent causes the release of TetR from TetO, thereby allowing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and has the chemical name 1-dimethylamino-2, 4a,5,7, 12-pentahydroxy-11-methyl-4, 6-di-oxo-1, 4a,11,11a,12,12 a-hexahydrotetracene-3-carboxamide.

In one embodiment, the codons of TetR are optimized for expression in mammalian cells (e.g., murine or human cells). Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, allowing for substantial changes in the nucleotide sequence of a given nucleic acid, without any changes in the amino acid sequence encoded by the nucleic acid. However, many organisms show differences in codon usage, which is also referred to as "codon bias" (i.e., bias in the usage of a particular codon for a given amino acid). Codon bias is often associated with the presence of a major species of tRNA for a particular codon, which in turn can increase the translation efficiency of the mRNA. Thus, coding sequences derived from a particular organism (e.g., prokaryotes) may be adjusted via codon optimization to improve expression in different organisms (e.g., eukaryotes).

Other particular variations of the Tet system include the following "Tet off" and "Tet on" systems. In the Tet-related system, transcription is inactive in the presence of Tc or Dox. In that system, a tetracycline-controlled transactivator protein (tTA), consisting of TetR fused to the strong transactivation domain from herpes simplex virus VP16, regulates expression of a target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). A TRE consists of a concatamer of TetO sequences fused to a promoter, typically the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate early promoter. In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and expression from the target gene remains inactive.

In contrast, in the Tet open system, transcription is active in the presence of Tc or Dox. The Tet-on system is based on a reverse tetracycline-controlled transactivator rtTA. Like tTA, rtTA is a fusion protein composed of TetR inhibitor and VP16 transactivation domain. However, four amino acid changes in the TetR DNA binding moiety alter the binding characteristics of rtTA such that it can only recognize the tetO sequence in the TRE of the target transgene in the presence of Dox. Thus, in the Tet-on system, transcription of TRE regulated target genes is stimulated by rtTA only in the presence of Dox.

Another inducible promoter system is the lac repressor system from e. (see Brown et al, Cell 49:603-612 (1987)). The lac repressor system functions by regulating transcription of a polynucleotide of interest operably linked to a promoter comprising a lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the polynucleotide of interest. Expression of the polynucleotide of interest is induced by a suitable inducer, such as isopropyl- β -D-thiogalactopyranoside (IPTG).

Another exemplary inducible promoter system for use in the present invention is the nuclear factor of activated T cells (NFAT) system. The NFAT transcription factor family is an important regulator of T cell activation. NFAT response elements are found, for example, in the IL-2 promoter (see, e.g., Durand, D. et al, Molecular. cell. biol.8,1715-1724 (1988); Clipsstone, NA, Crabtree, GR. Nature.1992357(6380): 695-7; Chmielewski, M. et al, Cancer research71.17(2011): 5697-. In some embodiments, an inducible promoter described herein comprises one or more (such as 2, 3,4, 5,6, or more) NFAT response elements. In some embodiments, the inducible promoter includes 6 NFAT responsive elements, e.g., including the nucleotide sequence of SEQ ID NO: 83. In some embodiments, an inducible promoter described herein comprises one or more (such as 2, 3,4, 5,6, or more) NFAT response elements linked to a minimal promoter (such as a minimal TA promoter). In some embodiments, the minimal TA promoter comprises the nucleotide sequence of SEQ ID NO 84. In some embodiments, the inducible promoter comprises the nucleotide sequence of SEQ ID NO 85.

To assess the expression of the polypeptide or portion thereof, the expression vector to be introduced into the cells may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a population of cells seeking to be transfected or infected with the viral vector. In other aspects, the selectable marker may be carried on a separate DNA segment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and analogs thereof.

The reporter gene is used to identify potential transfected cells and to evaluate the function of the regulatory sequences. In general, the reporter gene is a gene that is not present or expressed in the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some readily detectable property (e.g., enzymatic activity). Expression of the reporter gene is analyzed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may comprise genes encoding luciferase, β -galactose, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. In general, a construct with a minimal 5' flanking region displaying the highest expression level of a reporter gene was identified as a promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents that modulate the transcriptional capacity driven by the promoter.

In some embodiments, nucleic acids encoding a caTCR and/or a CSR according to any of the caTCR, CSR, and SSE described herein are provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding all of the polypeptide chains of the caTCR. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding all polypeptide chains of a CSR. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding all of the polypeptide chains of the caTCR and the CSR. In some embodiments, each of the one or more nucleic acid sequences is included in a separate vector. In some embodiments, at least some of the nucleic acid sequences are included in the same vector. In some embodiments, all nucleic acid sequences are included in the same vector. The vector may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors, such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

For example, in some embodiments, the caTCR is a dimer comprising a first caTCR polypeptide chain and a second caTCR polypeptide chain; and the CSR is a monomer comprising a single CSR polypeptide chain; and the nucleic acid comprises a first nucleic acid sequence encoding a first caTCR polypeptide chain, a second nucleic acid encoding a second caTCR chain, and a third nucleic acid sequence encoding a CSR polypeptide chain. In some embodiments, the first nucleic acid sequence is included in a first vector, the second nucleic acid sequence is included in a second vector, and the third nucleic acid sequence is included in a third vector. In some embodiments, the first and second nucleic acid sequences are included in a first vector and the third nucleic acid sequence is included in a second vector. In some embodiments, the first and third nucleic acid sequences are included in a first vector and the second nucleic acid sequence is included in a second vector. In some embodiments, the second and third nucleic acid sequences are included in a first vector and the first nucleic acid sequence is included in a second vector. In some embodiments, the first, second, and third nucleic acid sequences are included in the same vector. In some embodiments, the first nucleic acid sequence is under the control of a first promoter, the second nucleic acid sequence is under the control of a second promoter, and the third nucleic acid sequence is under the control of a third promoter. In some embodiments, some or all of the first, second, and third promoters have the same sequence. In some embodiments, some or all of the first, second, and third promoters have different sequences. In some embodiments, some or all of the first, second, and third nucleic acid sequences are expressed as a single transcript in a polycistronic vector under the control of a single promoter. See, e.g., Kim, JH et al, PLoSOne6(4): e18556,2011. In some embodiments, one or more of the promoters are inducible. In some embodiments, the third nucleic acid sequence encoding a CSR polypeptide chain is operably linked to an inducible promoter. In some embodiments, the inducible promoter includes one or more elements that respond to immune cell activation, such as NFAT responsive elements.

In some embodiments, some or all of the first, second, and third nucleic acid sequences have similar (such as substantially or about the same) expression levels in a host cell (such as a T cell). In some embodiments, some of the first, second, and third nucleic acid sequences have expression amounts that differ by at least about two-fold (such as at least about any of 2, 3,4, 5, or more fold) in a host cell (such as a T cell). Expression can be measured in mRNA or protein content. The expression amount of mRNA can be determined by measuring the amount of mRNA transcribed from a nucleic acid using various well-known methods including northern blotting, quantitative RT-PCR, microarray analysis, and the like. The amount of protein expression can be measured by known methods including immunocytochemical staining, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, luminescence analysis, mass spectrometry, high performance liquid chromatography, high pressure liquid chromatography-tandem mass spectrometry, and the like.

It is to be understood that features of the embodiments described herein can be adapted and combined to encompass embodiments comprising any number of nucleic acid sequences, e.g., wherein the nucleic acid encoding the calcr and/or CSR and/or SSE comprises five or more than five nucleic acid sequences (e.g., wherein the calcr and SSE each comprise 2 or more than 2 different polypeptide chains).

Thus, in some embodiments, there is provided a nucleic acid encoding: a) a dimeric caTCR comprising a first caTCR polypeptide chain and a second caTCR polypeptide chain according to any of the caTCR polypeptides described herein, the nucleic acid comprising i) a first caTCR nucleic acid sequence encoding the first caTCR polypeptide chain, and ii) a second caTCR nucleic acid sequence encoding the second caTCR polypeptide chain; and b) a monomeric CSR comprising a single CSR polypeptide chain according to any of the CSRs described herein, the nucleic acid further comprising a CSR nucleic acid sequence encoding a CSR polypeptide chain. In some embodiments, the first caTCR nucleic acid sequence is included in a first vector (such as a lentiviral vector), the second caTCR nucleic acid sequence is included in a second vector (such as a lentiviral vector), and the CSR nucleic acid sequence is included in a third vector (such as a lentiviral vector). In some embodiments, some or all of the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequences are included in the same vector (such as a lentiviral vector). In some embodiments, the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are each individually operably linked to a promoter. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the CSR nucleic acid sequence is operably linked to an inducible promoter. In some embodiments, the inducible promoter comprises one or more elements that respond to immune cell activation. In some embodiments, the CSR nucleic acid sequence is operably linked to an NFAT-derived promoter. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors).

In some embodiments, there is provided: a) a first vector (such as a lentiviral vector) comprising a nucleic acid encoding a dimeric calcr comprising a first calcr polypeptide chain and a second calcr polypeptide chain according to any one of the calcrs described herein, the nucleic acid comprising i) a first promoter operably linked to a first calcr nucleic acid sequence encoding the first calcr polypeptide chain; and ii) a second promoter operably linked to a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain; and b) a second vector (such as a lentiviral vector) comprising a nucleic acid encoding a monomeric CSR comprising a CSR polypeptide chain according to any of the CSRs described herein, the nucleic acid comprising a third promoter operably linked to a CSR nucleic acid sequence encoding a CSR polypeptide chain. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the first and/or second vector is a viral vector (such as a lentiviral vector).

In some embodiments, there is provided: a) a first vector (such as a lentiviral vector) comprising a nucleic acid encoding a dimeric calcr comprising a first calcr polypeptide chain and a second calcr polypeptide chain according to any one of the calcrs described herein, the nucleic acid comprising i) a first calcr nucleic acid sequence encoding a first calcr polypeptide chain; and ii) a caTCR nucleic acid sequence encoding a second caTCR polypeptide chain, wherein the first and second caTCR nucleic acid sequences are under the control of a first promoter; and b) a second vector (such as a lentiviral vector) comprising a nucleic acid encoding a monomeric CSR comprising a CSR polypeptide chain according to any of the CSRs described herein, the nucleic acid comprising a second promoter operably linked to a CSR nucleic acid sequence encoding a CSR polypeptide chain. In some embodiments, the first promoter is operably linked to the 5' end of the first catr nucleic acid sequence, and there is a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide, such as P2A, T2A, E2A, or F2A, that links the 3' end of the first catr nucleic acid sequence to the 5' end of the second catr nucleic acid sequence, wherein the first and second catr nucleic acid sequences are transcribed as a single RNA under the control of the first promoter. In some embodiments, the first promoter is operably linked to the 5' end of the second catr nucleic acid sequence, and there is a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide, such as P2A, T2A, E2A, or F2A, that links the 3' end of the second catr nucleic acid sequence to the 5' end of the first catr nucleic acid sequence, wherein the first and second catr nucleic acid sequences are transcribed as a single RNA under the control of the first promoter. In some embodiments, the first and/or second promoter is inducible. In some embodiments, the first and/or second vector is a viral vector (such as a lentiviral vector). It is understood that embodiments in which any of the nucleic acid sequences are exchanged, such as the first or second caTCR nucleic acid sequence is exchanged with a CSR nucleic acid sequence, are also contemplated.

In some embodiments, there is provided a vector (such as a viral vector, e.g., a lentiviral vector) comprising: a) a nucleic acid encoding a dimeric caTCR comprising a first caTCR polypeptide chain and a second caTCR polypeptide chain according to any of the caTCR polypeptides described herein, the nucleic acid comprising i) a first caTCR nucleic acid sequence encoding the first caTCR polypeptide chain; and ii) a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain; and b) a nucleic acid encoding a monomeric CSR comprising a CSR polypeptide chain according to any one of the CSRs described herein, the nucleic acid comprising a CSR nucleic acid sequence encoding a CSR polypeptide chain, wherein the first and second caTCR nucleic acid sequences are under the control of a first promoter, and wherein the CSR nucleic acid sequence is under the control of a second promoter. In some embodiments, the first promoter is operably linked to one of the caTCR nucleic acid sequences linked to the other caTCR nucleic acid sequence by a nucleic acid linker selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide, such as P2A, T2A, E2A, or F2A, such that the first and second caTCR nucleic acid sequences are transcribed into a single RNA under the control of the first promoter. In some embodiments, the first and/or second promoter is inducible. In some embodiments, the second promoter is an inducible promoter. In some embodiments, the inducible promoter comprises one or more elements that respond to immune cell activation. In some embodiments, the second promoter is an NFAT-derived promoter. In some embodiments, the NFAT-derived promoter comprises the nucleotide sequence of SEQ ID NO 85. In some embodiments, the vector is a viral vector (such as a lentiviral vector).

In some embodiments, there is provided a vector (such as a lentiviral vector) comprising: a) a nucleic acid encoding a dimeric caTCR comprising a first caTCR polypeptide chain and a second caTCR polypeptide chain according to any of the caTCR polypeptides described herein, the nucleic acid comprising i) a first caTCR nucleic acid sequence encoding the first caTCR polypeptide chain; and ii) a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain; and b) a nucleic acid encoding a monomeric CSR comprising a CSR polypeptide chain according to any one of the CSRs described herein, the nucleic acid comprising a CSR nucleic acid sequence encoding a CSR polypeptide chain, wherein the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are under the control of a single promoter. In some embodiments, the promoter is operably linked to one of the nucleic acid sequences linked to the other nucleic acid sequence by nucleic acid linkers individually selected from the group consisting of an Internal Ribosome Entry Site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide, such as P2A, T2A, E2A, or F2A, such that the first and second nucleic acid sequences and the CSR nucleic acid sequence are transcribed into a single RNA under the control of the promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the vector is a viral vector (such as a lentiviral vector).

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, for example, mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for making cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, the introduction of the polynucleotide into the host cell is performed by calcium phosphate transfection.

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors and particularly retroviral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses and adeno-associated viruses and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. It is contemplated that the nucleic acid is introduced into the host cell (in vitro, ex vivo, or in vivo) using a lipid formulation. In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule associated with the liposome and an oligonucleotide, encapsulated in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained in a lipid in suspension, contained or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, in the form of micelles or with a "collapsed" structure. It may also simply be interspersed into the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm, as well as classes of compounds that contain long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present invention, a variety of assays can be performed in order to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as southern and northern blots, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, identify reagents within the scope of the invention, for example, by immunological means (ELISA and western blotting) or by assays described herein.

Preparation of caTCR, CSR and SSE

In some embodiments, an antibody portion (e.g., Fab ', (Fab')2, Fv, or scFv) comprises a sequence derived from a monoclonal antibody according to any one of the cagcr, CSR, and SSE described herein comprising an antibody portion. In some embodiments, the antibody portion comprises a V from a monoclonal antibody_HAnd V_LA domain, or a variant thereof. In some embodiments, the antibody portion further comprises C from a monoclonal antibody _H1 and C_LA domain, or a variant thereof. Monoclonal antibodies can be prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature,256:495(1975) and Sergeeva et al, Blood,117(16): 4262-.

In the hybridoma method, a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to produce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro. The immunizing agent may comprise a fusion protein of the polypeptide or protein of interest, or a complex comprising at least two molecules, such as a complex comprising a peptide and an MHC protein. In general, if cells of human origin are desired, peripheral blood lymphocytes ("PBLs") are used; or spleen cells or lymph node cells if non-human mammalian origin is desired. The lymphocytes are then fused via an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press,1986), pages 59-103. Immortalized cell lines are typically transformed mammalian cells, specifically myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of cells lacking HGPRT.

In some embodiments, the immortalized cell lines fuse efficiently, support stable high-level expression of antibodies by the selected antibody-producing cells and are sensitive to a medium such as HAT medium. In some embodiments, the immortalized Cell line is a murine myeloma Cell line, which may be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. Kozbor, J.Immunol.,133:3001 (1984); brodeur et al, Monoclonal Antibody Production Techniques and applications (Marcel Dekker, Inc.: New York,1987) pp 51-63.

The culture medium in which the hybridoma cells are cultured can then be analyzed for the presence of monoclonal antibodies to the polypeptide. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Schacher analysis of Munson and Pollard, anal. biochem.,107:220 (1980).

After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and grown by standard methods. And (4) encoding. Suitable media for this purpose include, for example, Dulbecco's modified Eagle's Medium and RPMI-1640 Medium. Alternatively, the hybridoma cells can be grown in vivo in a mammal in the form of ascites.

Monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In some embodiments, the antibody portion comprises sequences from clones selected from a library of antibody portions (such as a phage library presenting scFv or Fab fragments), according to any of the cagcr, CSR, and SSE described herein comprising an antibody portion. Clones may be identified by screening combinatorial libraries for antibody fragments having a desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such Methods are described, for example, in Hoogenboom et al, Methods in Molecular Biology 178:1-37(O' Brien et al, eds., Human Press, Totowa, N.J.,2001), and are further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, Methods in Molecular Biology248:161-175(Lo eds., Human Press, Totowa, N.J., 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5): 1073-; fellouse, Proc.Natl.Acad.Sci.USA101(34): 12467-; and Lee et al, J.Immunol.Methods284(1-2):119-132 (2004).

In certain phage display methods, V_HAnd V_LThe lineage of the genes were cloned by Polymerase Chain Reaction (PCR) and recombined randomly in phage pools, respectively, which can then be screened for antigen binding phage as described in Winter et al, Ann. Rev. Immunol.12:433-455 (1994). Phage typically present antibody fragments in the form of single chain fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the native lineage can be cloned (e.g., from a human) to provide a single source of antibody against a wide range of non-self antigens as well as self antigens without any immunization as described by Griffiths et al, EMBO J,12: 725-. Finally, a native library can also be prepared synthetically as follows: cloning of unrearranged V gene segments from Stem cells and encoding CDR3 high Using PCR primers containing random sequencesVariable regions and the realization of in vitro rearrangements, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373 and U.S. patent publication nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936 and 2009/0002360.

Antibody portions can be prepared using phage display to screen antibody libraries specific for a target antigen, such as a peptide/MHC class I/II complex or a cell surface antigen. The library may be of at least 1 × 10⁹(such as at least about 1 × 10⁹、2.5×10⁹、5×10⁹、7.5×10⁹、1×10¹⁰、2.5×10¹⁰、5×10¹⁰、7.5×10¹⁰Or 1X 10¹¹Any of) a diverse repertoire of human scFv phage displays of unique human antibody fragments. In some embodiments, the library is a native human library constructed from DNA extracted from human PMBC and spleen from healthy donors, including all human heavy and light chain subfamilies. In some embodiments, the pool is a naive human pool constructed from DNA extracted from PBMCs isolated from patients with various diseases (such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases). In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is fully randomized, with all amino acids (except cysteine) being equally likely to be present at any given position (see, e.g., Hoet, R.M. et al, nat. Biotechnol.23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library is about 5 to about 24 (such as any one of about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) amino acids in length. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

Phage clones that bind to the target antigen with high affinity can be selected by: phage bind iteratively to target antigens that are bound to a solid support (such as for use in solution)Panning (elution) beads or mammalian cells for cell panning), followed by removal of non-bound phage and elution of specifically bound phage. In one example of solution panning, the target antigen may be biotinylated to be immobilized to a solid support. The biotin-labeled target antigen is mixed with a phage library and a solid support, such as streptavidin-conjugated dinonyl bead (Dynabead) M-280, and the target antigen-phage-bead complex is then isolated. The bound phage clones are then eluted and used to infect the appropriate host cell, such as E.coli XL1-Blue, for expression and purification. In the example of cell panning, T2 cells loaded with AFP peptide (TAP deficient, HLA-A02: 01)⁺Lymphoblastoid cell lines) are mixed with the phage library, after which the cells are collected and the bound clones are eluted and used to infect the appropriate host cells (for expression and purification). Multiple rounds of panning (such as any of about 2, 3,4, 5,6, or more) can be performed by solution panning, cell panning, or a combination of both to enrich for phage clones that specifically bind to the target antigen. The enriched phage clones can be tested for specific binding to the target antigen by any method known in the art, including, for example, ELISA and FACS.

Human and humanized antibody moieties

The calTCR, CSR, and SSE antibody portions can be human or humanized. The humanized form of a non-human (e.g., murine) antibody moiety is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (such as Fv, Fab ', F (ab')₂scFv, or other antigen binding subsequences of antibodies), typically contain minimal sequences derived from non-human immunoglobulins. Humanized antibody portions comprise human immunoglobulins, immunoglobulin chains or fragments thereof (recipient antibodies) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibody portions may also include residues not found in the recipient antibody portion, nor in the imported CDR or framework sequencesAnd (4) a base. In general, the humanized antibody portion may comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. See, e.g., Jones et al, Nature,321:522-525 (1986); riechmann et al, Nature,332: 323-E329 (1988); presta, curr, Op, struct, biol.,2: 593-.

In general, one or more amino acid residues from a non-human source have been introduced into the humanized antibody moiety. These non-human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable domain. According to some embodiments, humanization can be performed essentially following the method of Winter and colleagues (Jones et al, Nature,321:522-525 (1986); Riechmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536(1988)) by replacing the corresponding sequences of human antibodies with rodent CDRs or CDR sequences. Thus, such "humanized" antibody moieties are antibody moieties (U.S. Pat. No. 4,816,567) in which substantially less than the entire human variable domain has been substituted by the corresponding sequence from a non-human species. In fact, a humanized antibody moiety is typically a human antibody moiety in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

As an alternative to humanization, human antibody moieties may be produced. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of producing a complete human antibody repertoire without producing endogenous immunoglobulins following immunization. For example, homozygosis deletions of the antibody heavy chain Junction (JH) gene in chimeric and germline mutant mice have been described to result in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays to such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, PNAS USA,90:2551 (1993); jakobovits et al, Nature,362:255-258 (1993); bruggemann et al, Yeast in Immunol, 7:33 (1993); U.S. Pat. nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807 No; and WO 97/17852. Alternatively, human antibodies can be prepared by introducing a human immunoglobulin locus into a gene transfer product (e.g., a mouse in which endogenous immunoglobulin genes have been partially or completely inactivated). Following challenge, human antibody production was observed, which closely resembles that seen in humans in all respects, including gene rearrangement, combination, and antibody repertoire. Such methods are described, for example, in U.S. patent nos. 5,545,807; nos. 5,545,806; U.S. Pat. No. 5,569,825; 5,625,126 No; 5,633,425 No; and 5,661,016, and Marks et al, Bio/Technology,10:779-783 (1992); lonberg et al, Nature,368:856-859 (1994); morrison, Nature,368: 812-; fishwild et al, Nature Biotechnology,14: 845-; neuberger, Nature Biotechnology,14:826 (1996); lonberg and Huszar, Intern.Rev.Immunol.,13:65-93 (1995).

Human antibodies can also be produced by activated B cells in vitro (see U.S. Pat. nos. 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.mol.biol.,227:381 (1991); marks et al, J.mol.biol.,222:581 (1991). The techniques of Cole et al and Boerner et al can also be used to prepare human monoclonal antibodies. Cole et al, Monoclonal antibodies and Cancer Therapy, Alan R.Liss, page 77 (1985) and Boerner et al, J.Immunol.147 (1):86-95 (1991).

Other variants

In some embodiments, amino acid sequence variants of the antigen binding moieties provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antigen binding moiety. Amino acid sequence variants of the antigen binding moiety may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antigen binding moiety or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antigen binding moiety. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

In some embodiments, antigen binding module variants are provided having one or more amino acid substitutions. Sites of interest for substitutional mutation induction include the HVRs and FRs of the antibody portion. Amino acid substitutions can be introduced into the antigen binding module of interest, and the product screened for the desired activity, for example, to maintain/improve antigen binding or reduce immunogenicity.

Conservative substitutions are shown in table 4 below.

Table 4: conservative substitutions

Amino acids can be grouped into different classes according to common side chain properties:

a. hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

c. acidity: asp and Glu;

d. alkalinity: his, Lys, Arg;

e. residues that influence chain orientation: gly, Pro;

f. aromatic: trp, Tyr, Phe.

Non-conservative substitutions entail the exchange of a member of one of these classes for a member of the other class.

Exemplary substitutional variants are affinity matured antibody portions, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and variant antibody portions are presented on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) can be made in HVRs, for example, to improve antibody moiety affinity. Such alterations may be at HVR "hot spots" (i.e., residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196(2008)), and/or specificity decisionsIn fixed residues (SDR), wherein the resulting variants V are tested_HOr V_LBinding affinity of (4). The achievement of affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom et al Methods in molecular Biology 178:1-37(O' Brien et al eds., Human Press, Totowa, NJ, (2001)).

In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutation induction). A secondary library is then generated. The library is then screened to identify any antibody portion variants with the desired affinity. Another approach to introducing diversity involves HVR-guided pathways, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly commonly targeted.

In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such alterations do not substantially reduce the ability of the antibody portion to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be located outside of HVR "hotspots" or SDRs. Variants V provided hereinabove_HAnd V_LIn some embodiments of the sequences, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

One method suitable for identifying residues or regions of a mutation-inducing targetable antigen-binding moiety is termed "alanine scanning mutagenesis" and is described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antigen binding moiety with the antigen is affected. Other substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antigen binding moiety complex may be determined to identify the point of contact between the antigen binding moiety and the antigen. Such contact residues and adjacent residues may be targeted for or excluded from substitution candidates. Variants can be screened to determine if they contain the desired property.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions comprise an antigen-binding moiety with an N-terminal methionyl residue. Other insertional variants of the antigen-binding moiety comprise the fusion of the N-or C-terminus of the antigen-binding moiety with an enzyme (e.g. for ADEPT) or polypeptide that increases the serum half-life of the antigen-binding moiety.

Derivatives of the same

In some embodiments, a caTCR according to any of the catcrs described herein and/or a CSR according to any of the CSRs described herein and/or an SSE according to any of the SSEs described herein can be further modified to contain additional non-protein moieties known and readily available in the art. Suitable moieties for derivatizing the caTCR and/or CSR and/or SSE include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, polydextrose, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (either homopolymers or random copolymers), and polydextrose or poly (N-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polyoxypropylene/oxyethylene copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight, and may be branched or unbranched. The number of polymers attached to the caTCR and/or CSR and/or SSE can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to: the specific properties or functions of the calRs and/or CSRs and/or SSEs to be improved, regardless of whether the calRs and/or CSRs and/or SSE derivatives will be used in therapy or the like under defined conditions.

In some embodiments, conjugates of the caTCR and/or CSR and/or SSE and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In some embodiments, the non-protein moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein portion to a temperature that kills cells proximal to the caccr and/or CSR and/or SSE-non-protein portion.

Preparation of caTCR plus CSR immune cells

In one aspect, the invention provides an immune cell (such as a lymphocyte, e.g., a T cell) that expresses a caccr and CSR according to any of the embodiments described herein. Provided herein are exemplary methods of making an immune cell (such as a T cell) that expresses both calcr and CSR (a calcr plus CSR immune cell, such as a calcr plus CSR T cell).

In some embodiments, a calcr plus CSR immune cell (such as a calcr plus CSR T cell) can be generated by introducing one or more nucleic acids (including, e.g., a lentiviral vector) encoding a calcr (such as any of the calrs described herein) that specifically binds to a target antigen (such as a disease-associated antigen) and a CSR (such as any of the CSRs described herein) that specifically binds to a target ligand into the immune cell. Introduction of one or more nucleic acids into an immune cell can be achieved using techniques known in the art, such as those described herein for nucleic acids. In some embodiments, the cadcr plus CSR immune cells of the invention (such as cadcr plus CSR T cells) are capable of replicating in vivo, resulting in long-term persistence that can contribute to the sustained control of a disease associated with expression of a target antigen (such as cancer or a viral infection).

In some embodiments, the present invention relates to the administration of genetically modified T cells expressing a caTCR that specifically binds to a target antigen according to any of the catcrs described herein and a CSR that specifically binds to a target ligand according to any of the CSRs described herein using lymphocyte infusion for the treatment of a patient having or at risk of a disease and/or disorder associated with expression of a target antigen (also referred to herein as a "target antigen-positive" or "TA-positive" disease or disorder), including, for example, cancer or a viral infection. In some embodiments, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs are collected from a patient in need of treatment and T cells are activated and expanded using methods described herein and known in the art, and then infused back into the patient.

In some embodiments, a T cell (also referred to herein as a "calcr plus CSR T cell") is provided that expresses a calcr according to any of the calrs described herein that specifically binds to a target antigen and a CSR according to any of the CSRs described herein that specifically binds to a target ligand. The cadCR plus CSR T cells of the invention can undergo robust in vivo T cell expansion and can establish target antigen-specific memory cells that persist in blood and bone marrow at high levels for extended periods of time. In some embodiments, the cadcr plus CSR T cells of the invention infused into a patient can eliminate in vivo cells presenting a target antigen, such as cancer or virus-infected cells presenting the target antigen, in a patient with a target antigen-associated disease. In some embodiments, the cadcr plus CSR T cells of the invention infused into a patient can eliminate in vivo target antigen-presenting cells, such as target antigen-presenting cancer or virus-infected cells, in patients with target antigen-associated diseases that are difficult to treat with at least one conventional therapy.

Prior to T cell expansion and genetic modification, a source of T cells is obtained from an individual. 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 the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art may be used. In some embodiments of the invention, T cells may be obtained using any number of those of skill in the artKnown techniques (such as FICOLL)^TMIsolated) a blood unit collected from an individual. In some embodiments, the cells from the circulating blood of the individual are obtained by apheresis. The debridement product typically contains lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by the apheresis procedure may be washed to remove the plasma fraction and the cells placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. As the skilled person will readily appreciate, the washing step may be achieved by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g. Cobe 2991 cell processor, Baxter CytoMate or Haemonetics cell holder 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free²⁺No Mg²⁺PBS, PlasmaLyte a or other physiological saline solution with or without buffer. Alternatively, undesired components of the clearance sample can be removed and the cells resuspended directly in culture medium.

In some embodiments, by lysing red blood cells and, for example, by PERCOLL^TMT cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by depletion of monocytes by countercurrent centrifugal elutriation. Specific subpopulations of T cells (such as CD3)⁺、CD28⁺、CD4⁺、CD8⁺、CD45RA⁺And CD45RO⁺T cells) can be further isolated by positive or negative selection techniques. For example, in some embodiments, by beads (such as 3 × 28) bound with anti-CD 3/anti-CD 28 (i.e., 3 ×) s

M-450CD3/CD 28T) were incubated for a period of time sufficient for positive selection of the desired T cells to isolate the T cells. In some embodiments, the period of time is about 30 minutes. In some embodiments, the time period is 30 minutesRanging from hours to 36 hours or more (including all ranges between these values). In some embodiments, the period of time is at least 1, 2, 3,4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. To isolate T cells from patients with leukemia, cell yield can be increased using longer incubation times (such as 24 hours). Longer incubation times can be used to isolate T cells in any case where there are few T cells compared to other cell types, such as isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or immunocompromised individuals. In addition, the use of longer incubation times can improve the capture of CD8⁺Efficiency of T cells. Thus, by merely shortening or extending the time allowed for T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected with respect to or for culture initiation or other time points during the method. In addition, by increasing or decreasing the ratio of anti-CD 3 and/or anti-CD 28 antibodies on the beads or other surfaces, a subset of T cells can be preferentially selected for or against at the start of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present invention. In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "unselected" cells may also undergo other rounds of selection.

Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies to surface markers unique to the negatively selected cells. One method is to sort and/or select cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, monoclonal antibody cocktails typically comprise antibodies to CD 14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, it may be desirable to enrich for or positively select regulatory T cells that typically express CD4⁺、CD25⁺、CD62Lhi、GITR⁺And FoxP3⁺. OrIn some embodiments, the T regulatory cells are depleted by anti-CD 25 binding beads or other similar selection methods.

To isolate a desired cell population by positive or negative selection, the cell concentration and surface (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of the beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in some embodiments, a concentration of about 20 hundred million cells per milliliter is used. In some embodiments, a concentration of about 10 hundred million cells per milliliter is used. In some embodiments, greater than about 100,000,000 cells/ml are used. In some embodiments, a cell concentration of any of about 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, or 50,000,000 cells per milliliter is used. In some embodiments, a cell concentration of any of about 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, or 100,000,000 cells per milliliter is used. In some embodiments, a concentration of about 125,000,000 or about 150,000,000 cells/ml is used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest (such as CD 28-negative T cells) or from samples in which many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and will need to be obtained. For example, the use of high cell concentrations allows for more efficient selection of CD8, which typically has weaker CD28 expression⁺T cells.

In some embodiments of the invention, the T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, in particular after treatment with immune system-disrupting drugs, the quality of T cells obtained may be optimal or their ability to expand ex vivo shortly after treatment during the period when patients will normally recover from treatment. Likewise, enhanced transplantation and in vivo expansion of these cells may be in a preferred state after ex vivo manipulation using the methods described herein. Thus, the context of the present invention encompasses the collection of blood cells (including T cells), dendritic cells or other cells of hematopoietic lineage during this recovery phase. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and modulation protocols can be used to establish conditions in an individual, where it is desirable to repopulate, recycle, regenerate, and/or expand specific cell types, particularly during established time windows following treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Whether before or after genetically modifying the T cells to express the desired caTCR, CSR, and optionally SSE, the T cells can generally be activated or expanded using methods such as those described in: U.S. patent nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041 and U.S. patent application publication No. 20060121005.

In general, the T cells of the invention are expanded by contacting the surface to which they are attached with an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of the T cell. In particular, the population of T cells can be stimulated, such as by contact with an anti-CD 3 antibody or antigen-binding fragment thereof, or an anti-CD 2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryodin) bound to a calcium ionophore. To co-stimulate accessory molecules on the surface of T cells, ligands that bind accessory molecules are used. For example, a population of T cells can be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. To stimulate CD4⁺T cells or CD8⁺Proliferation of T cells, anti-CD 3 antibodies and anti-CD 28 antibodies. Examples of anti-CD 28 antibodies include 9.3, B-T3, XR-CD28(Diaclone,

france), may be as other parties as generally known in the artThe method used (Berg et al, transfer Proc.30(8):3975-3977, 1998; Haanen et al, J.Exp.Med.190(9):13191328,1999; Garland et al, J.Immunol meth.227(1-2):53-63,1999).

Genetic modification

In some embodiments, the cadcr plus CSR immune cells of the invention (such as cadcr plus CSR T cells) are produced by transducing immune cells (such as T cells prepared by the methods described herein) with one or more viral vectors encoding a cadcr as described herein and a CSR as described herein. Viral vector delivery systems include DNA and RNA viruses that have either an episomal or integrated genome after delivery to an immune cell. For a review of gene therapy programs, see Anderson, Science 256: 808-; nabel and Feigner, TIBTECH 11:211-217 (1993); mitani and Caskey, TIBTECH 11:162-166 (1993); dillon, TIBTECH 11: 167-; miller, Nature357:455-460 (1992); van Brunt, Biotechnology 6(10):1149-1154 (1988); vigne, reactive Neurology and Neuroscience 8:35-36 (1995); kremer and Perricaudet, British Medical Bulletin 51(l):31-44 (1995); and Yu et al, Gene Therapy 1:13-26 (1994). In some embodiments, the caTCR plus CSR immune cells comprise one or more vectors integrated into the genome of the caTCR plus CSR immune cells. In some embodiments, the one or more viral vectors are lentiviral vectors. In some embodiments, the caTCR plus CSR immune cells are caTCR plus CSR T cells that include a lentiviral vector integrated into their genome.

In some embodiments, the caTCR plus CSR immune cells are T cells modified to block or reduce expression of one or both of their endogenous TCR chains. For example, in some embodiments, the caTCR plus CSR immune cells are α β T cells modified to block or reduce expression of TCR α and/or β chains, or the caTCR plus CSR immune cells are γ δ T cells modified to block or reduce expression of TCR γ and/or δ chains. Cell modifications for disrupting gene expression include any such technique known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN-based gene knockout), and the like.

In some embodiments, a CRISPR/Cas system is used to produce caTCR plus CSR T cells with reduced expression of one or both of the endogenous TCR chains of the T cells. For a review of CRISPR/Cas systems for gene editing see, e.g., Jian W and Marraffini LA, annu. rev. microbiol.69, 2015; hsu PD et al, Cell,157(6), 1262-; and O' Connell MR et al, Nature 516: pp.263 and 266, 2014. In some embodiments, the TALEN-based genome editing is used to generate caTCR plus CSRT cells with reduced expression of one or both of the endogenous TCR chains of the T cells.

Enrichment of

In some embodiments, a method of enriching for caTCR plus CSR immune cells in a heterogeneous population of cells according to any of the caTCR plus CSR immune cells described herein is provided.

Specific subsets of caltcr plus CSR immune cells (such as calcr plus CSR T cells) that specifically bind to a target antigen and a target ligand can be enriched by positive selection techniques. For example, in some embodiments, the caTCR plus CSR immune cells (such as caTCR plus CSR T cells) are enriched by incubating the target antigen-bound beads and/or target ligand-bound beads for a period of time sufficient to positively select for the desired caTCR plus CSR immune cells. In some embodiments, the period of time is about 30 minutes. In some embodiments, the period of time is in the range of 30 minutes to 36 hours or more (including all ranges between these values). In some embodiments, the period of time is at least 1, 2, 3,4, 5, or 6 hours. In some embodiments, the period of time is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. With respect to the isolation of caTCR plus CSR immune cells present at low levels in heterogeneous cell populations, cell yield can be improved using longer incubation times (such as 24 hours). Longer incubation times can be used to isolate the caTCR plus CSR immune cells in any situation where there are fewer caTCR plus CSR immune cells than other cell types. Those skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present invention.

To isolate the desired population of caTCR plus CSR immune cells by positive selection, the cell concentration and surface (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of the beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact of the cells and beads. For example, in some embodiments, a concentration of about 20 hundred million cells per milliliter is used. In some embodiments, a concentration of about 10 hundred million cells per milliliter is used. In some embodiments, greater than about 100,000,000 cells/ml are used. In some embodiments, a cell concentration of any of about 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, or 50,000,000 cells per milliliter is used. In some embodiments, a cell concentration of any of about 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, or 100,000,000 cells per milliliter is used. In some embodiments, a concentration of about 125,000,000 or about 150,000,000 cells/ml is used. The use of high concentrations can result in increased cell yield, cell activation and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of the caTCR plus CSR immune cells that may weakly express caTCR and/or CSR.

In some of any such embodiments described herein, the enrichment results in minimal or substantially no depletion of the caTCR plus CSR immune cells. For example, in some embodiments, the enrichment results in depletion of less than about 50% (such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the caTCR plus CSR immune cells. Immune cell depletion can be determined by any means known in the art, including any means described herein.

In some of any such embodiments described herein, the enriching results in minimal or substantially no terminal differentiation of the caTCR plus CSR immune cells. For example, in some embodiments, enrichment results in less than about 50% (such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the caTCR plus CSR immune cells undergoing terminal differentiation. Immune cell differentiation can be determined by any means known in the art, including any means described herein.

In some of any such embodiments described herein, the enrichment results in minimal or substantially no internalization of the caTCR plus the caTCR and/or CSR on the CSR immune cells. For example, in some embodiments, enrichment results in internalization of less than about 50% (such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the caTCR and/or CSR on the caTCR plus CSR immune cells. Internalization of calcr and/or CSR on calcr plus CSR immune cells can be determined by any means known in the art, including any means described herein.

In some of any such embodiments described herein, the enrichment results in increased proliferation of the caTCR plus CSR immune cells. For example, in some embodiments, the enrichment results in an increase in the number of calcr plus CSR immune cells by at least about 10% (such as at least one of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000%, or more) after the enrichment.

Thus, in some embodiments, there is provided a method of enriching for a heterogeneous population of cells for cadr plus CSR immune cells expressing a cadr that specifically binds to a target antigen and a CSR that specifically binds to a target ligand, comprising: a) contacting a heterogeneous population of cells with a first molecule comprising a target antigen or one or more epitopes contained therein and/or a second molecule comprising a target ligand or one or more epitopes contained therein to form a complex comprising a cadcr plus CSR immune cell bound to the first molecule and/or a complex comprising a cadcr plus CSR immune cell bound to the second molecule; and b) isolating the complex from the heterogeneous cell population, thereby producing a cell population enriched for caTCR plus CSR immune cells. In some embodiments, the first and/or second molecules are individually immobilized to a solid support. In some embodiments, the solid support is a particulate (such as a bead). In some embodiments, the solid support is a surface (such as the bottom of a well). In some embodiments, the first and/or second molecules are individually labeled. In some embodiments, the label is a fluorescent molecule, an affinity label, or a magnetic label. In some embodiments, the method further comprises eluting the caTCR plus CSR immune cells from the first and/or second molecule and recovering the eluate.

Library screening

In some embodiments, to isolate a candidate caccr construct specific for a target antigen, a caccr library (e.g., cells expressing a nucleic acid library encoding a plurality of cacrs) can be exposed to a capture molecule comprising the target antigen or one or more epitopes contained therein, followed by isolation of an affinity member of the library that specifically binds to the capture molecule. In some embodiments, the capture molecule is immobilized on a solid support. In some embodiments, the support may be a bead surface, a microtiter plate, an immune tube, or any material known in the art to be suitable for such purposes. In some embodiments, the interaction with a labeled capture molecule (e.g., a biotin-labeled capture molecule) occurs in solution. In some embodiments, the procedure involves one or more washing steps (panning) for removing non-specific and non-reactive library members. In some embodiments, to purify the complex in solution, the complex is collected by immobilization or by centrifugation. In some embodiments, the affinity member is captured on a soluble biotin-labeled capture molecule, followed by immobilization of the affinity complex (affinity member and capture molecule) on streptavidin beads. In some embodiments, the solid support is a bead. In some embodiments, the beads comprise, for example, Magnetic beads (e.g., from Bangs Laboratories, Polysciences inc., Dynal Biotech, Miltenyi Biotech or Quantum Magnetic), non-Magnetic beads (e.g., Pierce and Upstatetechnology), monodisperse beads (e.g., Dynal Biotech and Microparticle Gmbh), and polydisperse beads (e.g., Chemagen). The use of magnetic beads is well described in the literature (Uhlen, M et al (1994) Advancin Biomagnetic Separation, BioTechniques Press, Westborough, Mass.). In some embodiments, the affinity member is purified by positive selection. In some embodiments, affinity members are purified by negative selection to remove undesirable library members. In some embodiments, affinity members are purified by positive and negative selection steps.

In some embodiments, to isolate a candidate CSR construct specific for a target ligand, a CSR library (e.g., a cell expressing a nucleic acid library encoding a plurality of CSRs) can be exposed to a capture molecule comprising the target ligand or one or more epitopes contained therein, followed by isolation of an affinity member of the library that specifically binds to the capture molecule. In some embodiments, the capture molecule is immobilized on a solid support. In some embodiments, the support may be a bead surface, a microtiter plate, an immune tube, or any material known in the art to be suitable for such purposes. In some embodiments, the interaction with a labeled capture molecule (e.g., a biotin-labeled capture molecule) occurs in solution. In some embodiments, the procedure involves one or more washing steps (panning) for removing non-specific and non-reactive library members. In some embodiments, to purify the complex in solution, the complex is collected by immobilization or by centrifugation. In some embodiments, the affinity member is captured on a soluble biotin-labeled capture molecule, followed by immobilization of the affinity complex (affinity member and capture molecule) on streptavidin beads. In some embodiments, the solid support is a bead. In some embodiments, the beads comprise, for example, Magnetic beads (e.g., from Bangs Laboratories, Polysciences inc., Dynal Biotech, miltenyi Biotech or Quantum Magnetic), non-Magnetic beads (e.g., Pierce and update technology), monodisperse beads (e.g., Dynal Biotech and Microparticle Gmbh), and polydisperse beads (e.g., Chemagen). In some embodiments, the affinity member is purified by positive selection. In some embodiments, affinity members are purified by negative selection to remove undesirable library members. In some embodiments, affinity members are purified by positive and negative selection steps.

In general, the techniques used to prepare the library constructs will be based on known genetic engineering techniques. In this regard, the nucleic acid sequence encoding the caTCR or CSR to be expressed in the library is incorporated into an expression vector appropriate for the type of expression system to be used. Suitable expression vectors for presentation in cells such as CD3+ cells are well known and described in the art. For example, in some embodiments, the expression vector is a viral vector, such as a lentiviral vector.

In some embodiments, a nucleic acid library is provided that includes sequences encoding a plurality of catrs according to any of the embodiments described herein. In some embodiments, the nucleic acid library comprises a viral vector encoding a plurality of catrs. In some embodiments, the viral vector is a lentiviral vector.

In some embodiments, there is provided a nucleic acid library comprising sequences encoding a plurality of CSRs according to any one of the embodiments described herein. In some embodiments, the nucleic acid library comprises viral vectors encoding multiple CSRs. In some embodiments, the viral vector is a lentiviral vector.

In some embodiments, there is provided a method of screening a library of nucleic acids according to any one of the embodiments described herein for sequences encoding a caTCR specific for a target antigen, comprising: a) introducing the nucleic acid pool into a plurality of cells such that the catrs are expressed on the surface on the plurality of cells; b) incubating a plurality of cells with a capture molecule comprising a target antigen or one or more epitopes contained therein; c) collecting the cells bound to the capture molecules; and d) isolating the sequences encoding the caTCR from the cells collected in step c), thereby identifying the caTCR specific for the target antigen. In some embodiments, the method further comprises one or more washing steps. In some embodiments, one or more washing steps are performed between steps b) and c). In some embodiments, the plurality of cells is a plurality of CD3+ cells. In some embodiments, the capture molecule is immobilized on a solid support. In some embodiments, the solid support is a bead. In some embodiments, collecting the cells bound to the capture molecules comprises eluting the cells from the capture ligand bound to the solid support and collecting the eluate. In some embodiments, the capture molecule is labeled with a label. In some embodiments, the label is a fluorescent molecule, an affinity label, or a magnetic label. In some embodiments, collecting the cells bound to the capture molecule comprises isolating a complex comprising the cells and the labeled ligand. In some embodiments, the cell is dissociated from the complex.

In some embodiments, there is provided a method of screening a nucleic acid library according to any of the embodiments described herein for sequences encoding CSRs specific for a target ligand comprising: a) introducing a nucleic acid pool into a plurality of cells such that the CSRs are expressed on the surface of the plurality of cells; b) incubating a plurality of cells with a capture molecule comprising a target ligand or one or more epitopes contained therein; c) collecting the cells bound to the capture molecules; and d) isolating sequences encoding CSR from the cells collected in step c), thereby identifying CSR specific for the target ligand. In some embodiments, the method further comprises one or more washing steps. In some embodiments, one or more washing steps are performed between steps b) and c). In some embodiments, the plurality of cells is a plurality of CD3+ cells. In some embodiments, the capture molecule is immobilized on a solid support. In some embodiments, the solid support is a bead. In some embodiments, collecting the cells bound to the capture molecules comprises eluting the cells from the capture ligand bound to the solid support and collecting the eluate. In some embodiments, the capture molecule is labeled with a label. In some embodiments, the label is a fluorescent molecule, an affinity label, or a magnetic label. In some embodiments, collecting the cells bound to the capture molecule comprises isolating a complex comprising the cells and the labeled ligand. In some embodiments, the cell is dissociated from the complex.

MHC proteins

MHC class I proteins are one of two major classes of Major Histocompatibility Complex (MHC) molecules (the other MHC class II) and are found on almost every nucleated cell of the body. Its function is to present intracellular protein fragments to T cells; healthy cells will be ignored and cells containing foreign or mutated proteins will be attacked by the immune system. Since MHC class I proteins present peptides derived from cytosolic proteins, the MHC class I presentation pathway is often referred to as the cytosolic or endogenous pathway. Class I MHC molecules bind peptides produced primarily by proteasome degradation of cytosolic proteins. The MHC I peptide complex is then inserted into the plasma membrane of the cell. The peptide binds to the extracellular portion of an MHC class I molecule. Thus, class I MHC functions to present intracellular proteins to cytotoxic T Cells (CTL). However, MHC class I can also present peptides generated from foreign proteins in a process known as cross-presentation.

MHC class I proteins are composed of two polypeptide chains: alpha and beta 2-microglobulin (. beta.2M). The two chains are non-covalently linked via interaction of β 2M with the α 3 domain. Only the alpha chain is polymorphic and encoded by the HLA gene, while the β 2M subunit is not polymorphic and is encoded by the β -2 microglobulin gene. The α 3 domain is the CD8 co-receptor that spans the plasma membrane and interacts with T cells. The α 3-CD8 interaction held the MHC I molecule in place, while the T Cell Receptor (TCR) on the surface of cytotoxic T cells bound to its α 1- α 2 heterodimer ligand, and the conjugated peptide was examined for antigenicity. The α 1 and α 2 domains fold to form a notch for peptide binding. MHC class I proteins bind peptides 8-10 amino acids in length.

MHC class II molecules are a family of molecules that are typically found only on antigen presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells. The antigens presented by class II peptides are derived from extracellular proteins (not cytosolic proteins as in class I); thus, the MHC class II dependent antigen presentation pathway is referred to as the endocytic or exogenous pathway. Loading of MHC class II molecules occurs by phagocytosis; extracellular proteins are endocytosed, digested in lysosomes, and the resulting epitope peptide fragments are loaded onto MHC class II molecules, which then migrate to the cell surface.

Like MHC class I molecules, class II molecules are also heterodimers, but in this case consist of two homogeneous peptides: alpha and beta chains. The sub-names α 1, α 2, etc. refer to individual domains within the HLA gene; each domain is typically encoded by a different exon within the gene, and some genes have other domains encoding leader sequences, transmembrane sequences, etc. Because the antigen binding groove of MHC class II molecules is open at both ends and the corresponding groove on class I molecules is closed at each end, the antigen presented by MHC class II molecules is longer, typically between 15 and 24 amino acid residues in length.

Human Leukocyte Antigen (HLA) genes are human versions of MHC genes. The three major MHC class I proteins in humans are HLA-A, HLA-B and HLA-C, and the 3 minor MHC class I proteins are HLA-E, HLA-F and HLA-G. The three major MHC class II proteins involved in antigen presentation in humans are HLA-DP, HLDA-DQ and HLA-DR, while the other MHC class II proteins HLA-DM and HLA-DO are involved in the internal processing and loading of antigens. HLA-A is listed as the gene with the fastest evolving coding sequence in humans. By 12 months 2013, there are 2432 known HLA-a alleles encoding 1740 active proteins and 117 null proteins. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger alpha chain component of HLA-A. Changes in the HLA-a α -chain are critical for HLA function. This variation promotes gene diversity in the population. Since each HLA has a different affinity for certain structures of the peptide, a larger number of HLA's means that more antigens are ' presented ' on the cell surface, increasing the likelihood that a subset of the population will be resistant to any given foreign invader. This reduces the likelihood that a single pathogen has the ability to destroy the entire human population. Each individual can express up to two types of HLA-A, each from its parent. Some individuals will inherit the same HLA-A from parents, reducing the individual HLA diversity; however, most individuals will receive two different copies of HLA-A. All HLA groups follow this same pattern. In other words, an individual may express only one or both of the 2432 known HLA-a alleles.

All alleles received at least four numerical classifications, e.g., HLA-A02: 12. A represents an HLA gene to which an allele belongs. There are many HLA-a alleles, simplifying classification by serotype. The next pair of numbers indicates this assignment. For example, HLA-a 02:02, HLA-a 02:04, and HLA-a 02:324 are all members of the a2 serotype (indicated by the a02 prefix). This group is the major factor responsible for HLA compatibility. All numbers thereafter were not determined by serotyping and were indicated via gene sequencing. The second set of numbers indicates which HLA proteins were produced. These were assigned in the order of discovery and by 12 months of 2013, there were 456 different known HLA-a02 proteins (assigned the designations HLA-a 02:01 to HLA-a 02: 456). The shortest HLA name possible contains both of these details. Each extension beyond this indicates a nucleotide change that may or may not alter the protein.

In some embodiments, the Fab-like antigen binding module specifically binds to a complex comprising a peptide derived from a disease-associated antigen (such as a tumor-associated or virus-encoded antigen) and an MHC class I protein, wherein the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G. In some embodiments, the MHC class I protein is HLA-A, HLA-B or HLA-C. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, the MHC class I protein is HLA-B. In some embodiments, the MHC class I protein is HLA-C. In some embodiments, the MHC class I protein is HLA-A01, HLA-A02, HLA-A03, HLA-A09, HLA-A10, HLA-A11, HLA-A19, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A34, HLA-A36, HLA-A43, HLA-A66, HLA-A68, HLA-A69, HLA-A74, or HLA-A80. In some embodiments, the MHC class I protein is HLA-A02. In some embodiments, the MHC class I protein is any one of HLA-A02: 01-555, such as HLA-A02: 01, HLA-A02: 02, HLA-A02: 03, HLA-A02: 04, HLA-A02: 05, HLA-A02: 06, HLA-A02: 07, HLA-A02: 08, HLA-A02: 09, HLA-A02: 10, HLA-A02: 11, HLA-A02: 12, HLA-A02: 13, HLA-A02: 14, HLA-A02: 15, HLA-A02: 16, HLA-A02: 17, HLA-A02: 18, HLA-A02: 19, HLA-A02: 20, HLA-A02: 21, HLA-A02: 22, or HLA-A24. In some embodiments, the MHC class I protein is HLA-a 02: 01. 39-46% of all caucasians (Caucasian) express HLA-A02: 01 and thus represent a suitable selection for MHC class I proteins for use in the present invention.

In some embodiments, the Fab-like antigen binding module specifically binds to a complex comprising a peptide derived from a disease-associated antigen (such as a tumor-associated or virus-encoded antigen) and an MHC class II protein, wherein the MHC class II protein is HLA-DP, HLA-DQ, or HLA-DR. In some embodiments, the MHC class II protein is HLA-DP. In some embodiments, the MHC class II protein is HLA-DQ. In some embodiments, the MHC class II protein is HLA-DR.

Peptides suitable for generating Fab-like antigen binding modules can be determined, for example, based on the presence of HLA (such as HLA-a 02:01) binding motifs and cleavage sites of proteasomes and immunoproteasomes using computer predictive models known to those of skill in the art. With respect to predicting MHC binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS17(12):1236-1237, 2001) and SYFPEITHI (see Schuler et al SYFPEITHI, Database forSearching and T-Cell Epitope prediction. Immunoformatory Methods in molecular Biology, Vol 409 (1):75-93,2007).

Once the appropriate peptide is identified, peptide synthesis can be accomplished according to protocols well known to those skilled in the art. The peptides of the invention, due to their relatively small size, can be synthesized directly in solution or on a solid support according to conventional peptide synthesis techniques. Various automated synthesizers are commercially available and can be used according to known protocols. Synthesis of peptides in solution phase has become a well-established procedure for large-scale production of synthetic peptides and is therefore a suitable alternative for the preparation of the peptides of the invention (see, e.g., Solid phase Peptide Synthesis, John Morow Stewart and Martin et al, Application of Almez-mediated Amidation reaction to liquid phase Peptide Synthesis, Tetrahedron Letters, Vol.39, p.1517-1520, 1998).

Pharmaceutical composition

Also provided herein are cadcr plus CSR immune cell compositions (such as pharmaceutical compositions also referred to herein as formulations) comprising immune cells (such as T cells) that present on their surface a cadcr according to any of the cadrs described herein and a CSR according to any of the CSRs described herein. In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

The composition may include: a homogeneous cell population comprising the same cell type of caltcr plus CSR immune cells and expressing the same caltcr and CSR, or a heterogeneous cell population comprising a plurality of caltcr plus CSR immune cell populations comprising different types of caltcr plus CSR immune cells, expressing different calcrs and/or expressing different CSRs. The composition can further include cells that are not a caTCR plus CSR immune cell.

Thus, in some embodiments, a cadcr plus CSR immune cell composition is provided that includes a homogeneous cell population of cadcr plus CSR immune cells (such as cadcr plus CSR T cells) of the same cell type and expresses the same cadcr and CSR. In some embodiments, the caTCR plus CSR immune cells are T cells. In some embodiments, the caTCR plus CSR immune cells are selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

In some embodiments, a cadcr plus CSR immune cell composition is provided that includes a heterogeneous cell population that includes a plurality of cadcr plus CSR immune cell populations comprising cadcr plus CSR immune cells of different cell types, expressing different cadrs, and/or expressing different CSRs. In some embodiments, the caTCR plus CSR immune cells are T cells. In some embodiments, each of the population of caTCR plus CSR immune cells is, independently of the other, a cell type selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, all of the caTCR plus CSR immune cells in the composition are of the same cell type (e.g., all of the caTCR plus CSR immune cells are cytotoxic T cells). In some embodiments, at least one of the cadcr plus CSR immune cell populations has a different cell type than the other cadcr plus CSR immune cell populations (e.g., one cadcr plus CSR immune cell population consists of cytotoxic T cells and another cadcr plus CSR immune cell population consists of natural killer T cells). In some embodiments, each population of caTCR plus CSR immune cells expresses the same caTCR. In some embodiments, at least one of the cadcr plus CSR immune cell populations expresses a cadcr that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each of the cadcr plus CSR immune cell populations expresses a cadcr that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to the same target antigen. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a cadcr that specifically binds to a target antigen that is different from the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a different target antigen, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a target antigen associated with the same disease or disorder (e.g., each target antigen is associated with a cancer such as breast cancer). In some embodiments, each of the population of caTCR plus CSR immune cells expresses the same CSR. In some embodiments, at least one of the cadcr plus CSR immune cell populations expresses a CSR that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each of the population of caTCR plus CSR immune cells expresses a CSR that is different from the other population of caTCR plus CSR immune cells in some embodiments, each of the population of caTCR plus CSR immune cells expresses a CSR that specifically binds to the same target ligand. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a CSR that specifically binds to a different target ligand than the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses CSRs that specifically bind to different target ligands, each population of caTCR plus CSR immune cells expresses CSRs that specifically bind to target ligands associated with the same disease or disorder (e.g., each target ligand is associated with a cancer such as breast cancer). In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

Thus, in some embodiments, there is provided a cadcr plus CSR immune cell composition comprising a plurality of cadcr plus CSR immune cell populations according to any one of the embodiments described herein, wherein all the cadcr plus CSR immune cells in the composition are of the same cell type (e.g., all the cadcr plus CSR immune cells are cytotoxic T cells), and wherein each cadr plus CSR immune cell population expresses a cadcr that is different from the other cadr plus CSR immune cell populations. In some embodiments, the caTCR plus CSR immune cells are T cells. In some embodiments, the caTCR plus CSR immune cells are selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to the same target antigen. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a cadcr that specifically binds to a target antigen that is different from the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a different target antigen, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a target antigen associated with the same disease or disorder (e.g., each target antigen is associated with a cancer such as breast cancer). In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

In some embodiments, there is provided a cadcr plus CSR immune cell composition comprising a plurality of cadcr plus CSR immune cell populations according to any one of the embodiments described herein, wherein all of the cadcr plus CSR immune cells in the composition are of the same cell type (e.g., all of the cadcr plus CSR immune cells are cytotoxic T cells), and wherein each cadcr plus CSR immune cell population expresses a cadcr that is different from the other cadcr plus CSR immune cell populations. In some embodiments, the caTCR plus CSR immune cells are T cells. In some embodiments, the caTCR plus CSR immune cells are selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each of the population of caTCR plus CSR immune cells expresses a CSR that specifically binds to the same target ligand. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a CSR that specifically binds to a different target ligand than the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses CSRs that specifically bind to different target ligands, each population of caTCR plus CSR immune cells expresses CSRs that specifically bind to target ligands associated with the same disease or disorder (e.g., each target ligand is associated with a cancer such as breast cancer). In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

In some embodiments, a composition is provided that includes a plurality of calcr plus CSR immune cell populations according to any of the embodiments described herein, wherein at least one of the calcr plus CSR immune cell populations has a different cell type than the other calcr plus CSR immune cell populations. In some embodiments, all of the population of caTCR plus CSR immune cells have different cell types. In some embodiments, the caTCR plus CSR immune cells are T cells. In some embodiments, each of the population of caTCR plus CSR immune cells is, independently of the other, a cell type selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, each population of caTCR plus CSR immune cells expresses the same caTCR. In some embodiments, at least one of the cadcr plus CSR immune cell populations expresses a cadcr that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each of the cadcr plus CSR immune cell populations expresses a cadcr that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to the same target antigen. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a cadcr that specifically binds to a target antigen that is different from the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a different target antigen, each population of caTCR plus CSR immune cells expresses a caTCR that specifically binds to a target antigen associated with the same disease or disorder (e.g., each target antigen is associated with a cancer such as breast cancer). In some embodiments, each of the population of caTCR plus CSR immune cells expresses the same CSR. In some embodiments, at least one of the cadcr plus CSR immune cell populations expresses a CSR that is different from the other cadcr plus CSR immune cell populations. In some embodiments, each of the population of caTCR plus CSR immune cells expresses a CSR that is different from the other population of caTCR plus CSR immune cells in some embodiments, each of the population of caTCR plus CSR immune cells expresses a CSR that specifically binds to the same target ligand. In some embodiments, at least one of the population of cadcr plus CSR immune cells expresses a CSR that specifically binds to a different target ligand than the other population of cadcr plus CSR immune cells (e.g., one population of cadcr plus CSR immune cells specifically binds to a pMHC complex and another population of cadcr plus CSR immune cells specifically binds to a cell surface receptor). In some embodiments, where at least one of the population of caTCR plus CSR immune cells expresses CSRs that specifically bind to different target ligands, each population of caTCR plus CSR immune cells expresses CSRs that specifically bind to target ligands associated with the same disease or disorder (e.g., each target ligand is associated with a cancer such as breast cancer). In some embodiments, the caTCR plus CSR immune cell composition is a pharmaceutical composition.

At various times during the preparation of the composition, it may be desirable or advisable to cryopreserve the cells. The terms "freezing" and "cryopreservation" are used interchangeably. Freezing comprises freeze-drying.

As understood by one of ordinary skill, frozen cells can be destructive (see Mazur, P.,1977, Cryobiology 14:251- & 272), but there are a number of procedures available to prevent such destruction. For example, damage may be avoided by (a) using a cryoprotectant, (b) controlling the freezing rate, and/or (c) storing at a temperature low enough to minimize degradation reactions. Exemplary cryoprotectants include Dimethylsulfoxide (DMSO) (Lovelock and Bishop,1959, Nature 183:1394-, llbery eds, Butterworth, London, page 59). In particular embodiments, DMSO may be used. The protective effect of DMSO can be potentiated by the addition of plasma (e.g., to a concentration of 20-25%). After addition of DMSO, cells can be kept at 0 ℃ until frozen, as a 1% DMSO concentration can be toxic at temperatures above 4 ℃.

In cryopreservation of cells, slowly controlled cooling rates can be critical and different cryoprotectants (Rapatz et al, 1968, Cryobiology 5(1):18-25) and different cell types have different optimal cooling rates (see, for example, Rowe and Rinfret,1962, Blood 20: 636; Rowe,1966, Cryobiology 3(1): 12-18; Lewis et al, 1967, Transfusion7(1): 17-32; and Mazur,1970, Science 168: 939-. The amount of heat in the melting phase, where water becomes ice, should be minimal. The cooling procedure may be performed by using, for example, a programmable freezer or a methanol bath procedure. The programmable refrigeration device allows determination of the optimal cooling rate and enables standard renewable cooling.

In particular embodiments, DMSO-treated cells may be pre-cooled on ice and transferred to a tray containing chilled methanol, which in turn is placed in a mechanical refrigerator (e.g., Harris or Revco) at-80 ℃. Thermocouple measurements of the methanol bath and the sample indicate that a cooling rate of 1 ℃ to 3 ℃/minute may be preferred. After at least two hours, the sample may reach a temperature of-80 ℃ and may be placed directly in liquid nitrogen (-196 ℃).

After thorough freezing, the cells can be quickly transferred to a long-term cryogenic storage container. In a preferred embodiment, the sample may be stored cryogenically in liquid nitrogen (-196 ℃) or in vapor (-1 ℃). The availability of a high efficiency liquid nitrogen refrigerator facilitates such storage.

Other considerations and procedures regarding handling, cryopreservation and long term storage of cells can be found in the following exemplary references: U.S. patent nos. 4,199,022, 3,753,357 and 4,559,298; gorin,1986, Clinics In Haematology 15(1): 19-48; Bone-Marrow Conservation, Culture and Transmission, Proceedings of a Panel, Moscow, 7.7.22-26.1968, International atomic Energy Agency, Vienna, page 107-186; livesey and Linner,1987, Nature 327: 255; linner et al, 1986, J.Histochem.Cytochem.34(9): 1123-1135; simione,1992, J.Parenter.Sci.Technol.46(6): 226-32.

After cryopreservation, the frozen cells can be thawed for use according to methods known to the skilled artisan. The frozen cells are preferably thawed quickly and cryopreserved immediately after thawing. In a particular embodiment, the vial containing the frozen cells may be immersed in a warm water bath up to its neck; gentle rotation will ensure that the cell suspension mixes as it thaws and improves heat transfer from the warm water to the internal ice cubes. Once the ice has completely melted, the vial can be placed on the ice immediately.

In particular embodiments, methods of preventing cell agglutination may be used during thawing. An exemplary method includes: deoxyribonuclease (Spitzer et al, 1980, Cancer 45:3075-3085), low molecular weight polydextrose and citrate, hydroxyethyl starch (Stiff et al, 1983, Cryobiology 20:17-24), and the like are added before and/or after freezing. As understood by the skilled person, if a cryoprotectant toxic to humans is used, it should be removed prior to therapeutic use. DMSO has no serious toxicity.

Exemplary vehicles and modes of cell administration are described in U.S. patent publication No. 2010/0183564, pages 14-15. Other pharmaceutical carriers are described in Remington, The Science and Practice of Pharmacy, 21 st edition, David B.Troy, eds, Lippicott Williams & Wilkins (2005).

In particular embodiments, the cells can be collected from the culture medium, washed and concentrated in a carrier to a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks 'solution, Ringer's solution, Nonnosol-R (Abbott labs), Plasma-Lite A (R) (Baxter laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

In particular embodiments, the carrier can be supplemented with Human Serum Albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, the infusion vehicle comprises buffered saline with 5% HAS or dextrose. Other isotonic agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

The carrier may comprise a buffer, such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, and/or a trimethylamine salt.

Stabilizers refer to a wide range of excipients that can range in function from bulking agents to additives that help prevent cell adhesion to the container wall. Typical stabilizers may comprise polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, and cyclic alcohols such as inositol; PEG; an amino acid polymer; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; and polysaccharides such as polydextrose.

Where desired or where advantageous, the compositions may contain a local anaesthetic such as lidocaine (lidocaine) to reduce pain at the site of injection.

Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methyl paraben, propyl paraben, octadecyl dimethyl benzalkonium chloride, benzalkonium halides, hexakis ammonium chloride, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

A therapeutically effective amount of cells within a composition can be greater than 10²One cell, greater than 10³One cell, greater than 10⁴One cell, greater than 10⁵One cell, greater than 10⁶One cell, greater than 10⁷One cell, greater than 10⁸One cell, greater than 10⁹One cell, greater than 10¹⁰Single cell or greater than 10¹¹And (4) cells.

In the compositions and formulations disclosed herein, the volume of cells is typically one liter or less, 500ml or less, 250ml or less, or 100ml or less. Thus, the cell density administered is typically greater than 10⁴Cell/ml, 10⁷Cell/ml or 10⁸Individual cells/ml.

Also provided herein are nucleic acid compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising any of the nucleic acids encoding the cacrs and/or CSRs and/or SSEs described herein. In some embodiments, the nucleic acid composition is a pharmaceutical composition. In some embodiments, the nucleic acid composition further comprises any one of: isotonic agents, excipients, diluents, thickeners, stabilizers, buffers and/or preservatives; and/or an aqueous vehicle such as purified water, an aqueous sugar solution, a buffer solution, physiological saline, an aqueous polymer solution, or water free of ribonuclease. The amounts of these additives and aqueous vehicle to be added may be appropriately selected depending on the form of use of the nucleic acid composition.

The compositions and formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage. The compositions and formulations may further be formulated for injection in the bone marrow, intravenously, intradermally, intraarterially, intratubercularly, intralymphatically, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, rectally, topically, intrathecally, intratumorally, intramuscularly, intravesicularly, and/or subcutaneously.

Formulations for in vivo administration must be sterile. This is readily achieved by filtration, for example, through sterile filtration membranes.

Methods of treatment using caTCR plus CSR immune cells

The cadcr plus CSR immune cells of the invention can be administered to a subject (e.g., a mammal such as a human) to treat a disease and/or disorder associated with expression of a Target Antigen (TA) (also referred to herein as a "target antigen positive" or "TA positive" disease or disorder), including, for example, cancer and infectious diseases (such as viral infections). Accordingly, the present application provides in some embodiments a method for treating a target antigen-positive disease (such as cancer or a viral infection) in an individual, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising a caccr plus CSR immune cell according to any of the embodiments described herein. In some embodiments, the cancer is selected from, for example, the group consisting of: adrenocortical carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, bile duct carcinoma, colorectal carcinoma, esophageal carcinoma, glioblastoma, glioma, hepatocellular carcinoma, head and neck carcinoma, renal carcinoma, leukemia, lung carcinoma, lymphoma, melanoma, mesothelioma, multiple myeloma, pancreatic carcinoma, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian carcinoma, prostate carcinoma, sarcoma, gastric carcinoma, uterine carcinoma and thyroid carcinoma. In some embodiments, the viral infection is caused by a virus selected from the group consisting of, for example: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Kaposi's Sarcoma-associated herpes Virus (KSHV), Human Papilloma Virus (HPV), Molluscum Contagiosum Virus (MCV), Human T-cell leukemia Virus 1(Human T cell leukemia Virus 1, HTLV-1), Human Immunodeficiency Virus (HIV), and Hepatitis C Virus (HCV).

For example, in some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR comprising i) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of the TCR, and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and ii) an antigen binding moiety that specifically binds to a target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an intracellular signaling domain capable of providing a costimulatory signal to an immune cell. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the TCR is an α β TCR, and the first and second TCR-TMs are derived from TCR α and β subunit transmembrane domains. In some embodiments, the TCR is a γ δ TCR, and the first and second TCR-TMs are derived from TCR γ and δ subunit transmembrane domains. In some embodiments, the first TCRD further comprises a first TCR-binding peptide or fragment thereof, and/or the second TCRD further comprises a second TCR-binding peptide or fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR endodomain, and/or the second TCRD further comprises a second TCR endodomain. In some embodiments, the first TCR endodomain comprises a sequence from an endodomain of a TCR subunit from which the first TCR-TM is derived, and/or the second TCR endodomainIncluding sequences from the intracellular domain of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the caTCR further comprises at least one accessory endodomain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD 40). In some embodiments, the calcr further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have a binding affinity for each other that stabilizes the calcr. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C)_H1 and C_LAntibody domain) or a variant thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3 δ epsilon, CD3 γ epsilon, and ζ ζ ζ. In some embodiments, the TCRM allows for enhanced recruitment of at least one TCR-associated signaling molecule as compared to a TCRM comprising a T cell receptor transmembrane domain. In some embodiments, the TCRM promotes the formation of the caTCR-CD3 complex. In some embodiments, there is a spacer moiety between any two caTCR moieties or domains. In some embodiments, the antigen binding moiety is an antibody moiety. In some embodiments, the antibody moiety is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the antigen binding moiety is multispecific (e.g., bispecific). In some embodiments, the target antigen is a cell surface antigen. In some embodiments, the cell surface antigen is selected from the group consisting of proteins, carbohydrates, and lipids. In some embodiments, the cell surface antigen is a disease-associated antigen, such as a tumor-associated or virus-encoded antigen. In some embodiments, the cell surface antigen is CD19, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the target antigen is a surface-presented peptide/MHC complex. In some embodiments, the peptide/MHC complex includes peptides derived from a disease-associated antigen (such as a tumor-associated or virus-encoded antigen) and an MHC protein. In some embodiments, the peptidethe/MHC complex includes peptides and MHC proteins, wherein the peptides are derived from a protein selected from the group consisting of WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the MHC protein is an MHC class I protein. In some embodiments, the MHC class I protein is HLA-A. In some embodiments, HLA-A is HLA-A02. In some embodiments, the HLA-a02 is HLA-a x 02: 01. In some embodiments, the target ligand of CSR is a cell surface antigen. In some embodiments, the target ligand is a peptide/MHC complex. In some embodiments, the target ligand of the CSR is the same as the target antigen of the caTCR. In some embodiments, the target ligand is different from the target antigen. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is, e.g., CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL 5. In some embodiments, the cancer-associated ligand is a peptide/MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule comprises PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL 9. In some embodiments, the target ligand is an apoptotic molecule. In some embodiments, the apoptotic molecule comprises FasL, FasR, TNFR1, and TNFR 2. In some embodiments, the ligand binding domain is an antibody moiety. In some embodiments, the ligand binding domain antibody portion is a Fab, Fab ', (Fab')2, Fv, or single chain Fv (scfv). In some embodiments, the ligand binding domain is all or a portion of an extracellular domain of a receptor of (or derived from) a target ligand. In some embodiments, receptors include, for example, FasR, TNFR1, TNFR2, PD-1, CD28, CTLA-4, ICOS, BTLA, KIR, LAG-3, 4-1BB, OX40, CD27, and TIM-3. In some embodiments, the transmembrane domain of a CSR comprises a transmembrane domain derived from, for example, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154. In some embodiments, synergistic stimulation of CSRThe stimulatory domain comprises, consists essentially of, or consists of all or a portion of the intracellular domain of an immune cell costimulatory molecule comprising, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the CSR further comprises a spacer domain between any of the ligand binding domain, transmembrane domain, and costimulatory signaling domain. In some embodiments, the spacer domain comprises a peptide linker connecting the two CSR domains. In some embodiments, the immune cell is a γ δ T cell. In some embodiments, the immune cell is an α β T cell modified to block or reduce expression of TCR α and/or β chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR that specifically binds a target antigen, the caTCR comprising i) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring α β TCR, and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring α β TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and ii) an antigen binding moiety that specifically binds to the target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an intracellular signaling domain capable of providing a costimulatory signal to an immune cell.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR that specifically binds a target antigen, the caTCR comprising i) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring γ δ TCR, and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring γ δ TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and ii) an antigen binding moiety that specifically binds to the target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an intracellular signaling domain capable of providing a costimulatory signal to an immune cell.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a calTCR that specifically binds a target antigen, the calTCR comprising i) a first TCRD comprising a first TCR-TM derived from the amino acid sequence of SEQ ID NO. 5, and a second TCRD comprising a second TCR-TM derived from the amino acid sequence of SEQ ID NO. 6, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and b) an antigen binding moiety that specifically binds to the target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an intracellular signaling domain capable of providing a costimulatory signal to an immune cell. In some embodiments, at least one of the first TCR-TM comprises one or more (such as 2, 3,4, 5, or more) amino acid substitutions as compared to the amino acid sequence from which it is derived. In some embodiments, each TCR-TM includes one or more (such as 2, 3,4, 5, or more) amino acid substitutions independently of the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each TCR-TM comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a calTCR that specifically binds a target antigen, the calTCR comprising i) a first TCRD comprising a first TCR-TM derived from the amino acid sequence of SEQ ID NO. 7, and a second TCRD comprising a second TCR-TM derived from the amino acid sequence of SEQ ID NO. 8, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and b) an antigen binding moiety that specifically binds to the target antigen, wherein the antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an intracellular signaling domain capable of providing a costimulatory signal to an immune cell. In some embodiments, at least one of the first TCR-TM comprises one or more (such as 2, 3,4, 5, or more) amino acid substitutions as compared to the amino acid sequence from which it is derived. In some embodiments, each TCR-TM includes one or more (such as 2, 3,4, 5, or more) amino acid substitutions independently of the amino acid sequence from which it is derived. In some embodiments, the first TCR-TM and/or the second TCR-TM each, independently of the other, comprise no more than 5 amino acid substitutions as compared to the amino acid sequence from which they are derived. In some embodiments, at least one of the TCR-TMs comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, each TCR-TM comprises a single amino acid substitution compared to the amino acid sequence from which it is derived. In some embodiments, at least one of the substituted amino acids in the first TCR-TM is positioned such that it can interact with at least one of the substituted amino acids in the second TCR-TM in the caTCR. In some embodiments, the first TCR-TM and the second TCR-TM are selected according to any one of the caTCRs listed in Table 2. In some embodiments, the CSR domain is selected according to any one of the CSRs listed in table 3.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR that specifically binds a target antigen, the caTCR comprising: i) a first TCRD comprising a first TCR-TM having the amino acid sequence of SEQ ID No. 10, and a second TCRD comprising a second TCR-TM having the amino acid sequence of SEQ ID No. 16, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule, and b) an antigen binding moiety that specifically binds to a target antigen, wherein said antigen binding moiety is linked to the first and/or second TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a ligand binding domain that specifically binds to a target ligand, ii) a transmembrane domain, and iii) an immune cell costimulatory molecule fragment (fCSM) comprising an intracellular signaling domain capable of providing a costimulatory signal to an immune cell, wherein the costimulatory molecule fragment comprises the amino acid sequence of any one of SEQ ID NOs 51-56 and 86-89. In some embodiments, the CSR transmembrane domain is derived from CD 8. In some embodiments, the CSR comprises a transmembrane protein fragment (fTMP), wherein the transmembrane protein fragment comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the CSR comprises a spacer peptide following the ligand binding domain. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO 103 or 104. In some embodiments, the CSR domain is according to any of the CSRs listed in table 3One is selected. In some embodiments, the target antigen is CD 19. In some embodiments, the target ligand is CD 19. In some embodiments, the caTCR antigen-binding moiety is a Fab-like antigen-binding moiety comprising a V comprising the amino acid sequence of SEQ ID NO:58_HDomains and V comprising the amino acid sequence of SEQ ID NO 59_LA domain. In some embodiments, the target antigen is CD19 and the target ligand is CD 19. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ id No. 77. In some embodiments, the target antigen is CD19 and the target ligand is CD 20. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the target antigen is an AFP peptide/MHC complex (e.g., an AFP158/HLA-a02 complex) and the target ligand is GPC 3. In some embodiments, the caTCR antigen-binding moiety is a Fab-like antigen-binding moiety comprising a V comprising the amino acid sequence of SEQ ID NO:62_HDomain and V comprising the amino acid sequence of SEQ ID NO 63_LA domain. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 79.

In some embodiments, there is provided a method of treating a CD 19-associated disease (such as cancer) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR that specifically binds CD19, the caTCR comprising i) a first TCRD comprising a first TCR-TM having the amino acid sequence of SEQ ID NO. 10, and a second TCRD comprising a second TCR-TM having the amino acid sequence of SEQ ID NO. 16, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) a Fab-like antigen binding module comprising a first Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO:58_HA domain, and a second Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO 59_LA domain wherein the first Fab chain is linked to one of the first and second TCRDs and the second Fab chain is linked to the other TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) an scFv that specifically binds to CD19, saidscFv includes V comprising the amino acid sequence of SEQ ID NO:58_HDomains and V comprising the amino acid sequence of SEQ ID NO 59_LA domain; ii) a transmembrane domain, and iii) an immune cell costimulatory molecule fragment (fCSM) comprising an intracellular signaling domain capable of providing a costimulatory signal to an immune cell, wherein the costimulatory molecule fragment comprises the amino acid sequence of any one of SEQ ID NOs 51-56 and 86-89. In some embodiments, the CSR transmembrane domain is derived from CD 8. In some embodiments, the CSR comprises a transmembrane protein fragment (fTMP), wherein the transmembrane protein fragment comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the CSR comprises a spacer peptide following the ligand binding domain. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO 103 or 104. In some embodiments, the CSR domain is selected according to any one of the CSRs listed in table 3. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ ID NO 77. In some embodiments, the CD 19-related disease is leukemia, such as Acute Lymphoblastic Leukemia (ALL).

In some embodiments, there is provided a method of treating a CD19/CD 20-associated disease (such as cancer) in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a caTCR that specifically binds CD19, the caTCR comprising i) a first TCRD comprising a first TCR-TM having the amino acid sequence of SEQ ID NO. 10, and a second TCRD comprising a second TCR-TM having the amino acid sequence of SEQ ID NO. 16, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) a Fab-like antigen binding module comprising a first Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO:58_HA domain, and a second Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO 59_LA domain wherein the first Fab chain is linked to one of the first and second TCRDs and the second Fab chain is linked to the other TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a scFv that specifically binds to CD20, said scFv comprising ammonia comprising SEQ ID NO:60V of an amino acid sequence_HDomain and V comprising the amino acid sequence of SEQ ID NO 61_LA domain; ii) a transmembrane domain, and iii) an immune cell costimulatory molecule fragment (fCSM) comprising an intracellular signaling domain capable of providing a costimulatory signal to an immune cell, wherein the costimulatory molecule fragment comprises the amino acid sequence of any one of SEQ ID NOs 51-56 and 86-89. In some embodiments, the CSR transmembrane domain is derived from CD 8. In some embodiments, the CSR comprises a transmembrane protein fragment (fTMP), wherein the transmembrane protein fragment comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the CSR comprises a spacer peptide following the ligand binding domain. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO 103 or 104. In some embodiments, the CSR domain is selected according to any one of the CSRs listed in table 3. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 81.

In some embodiments, there is provided a method of treating an AFP/GPC 3-related disease (such as cancer, e.g., liver cancer) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising immune cells (such as T cells or natural killer cells) that present on their surface: a) a calTCR that specifically binds an AFP peptide/MHC complex (e.g., the AFP158/HLA-A02 complex) comprising i) a first TCRD comprising a first TCR-TM having the amino acid sequence of SEQ ID NO:10 and a second TCRD comprising a second TCR-TM having the amino acid sequence of SEQ ID NO:16, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-related signaling molecule; and b) a Fab-like antigen binding module comprising a first Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO:62_HA domain, and a second Fab chain comprising a V comprising the amino acid sequence of SEQ ID NO 63_LA domain wherein the first Fab chain is linked to one of the first and second TCRDs and the second Fab chain is linked to the other TCRD; and b) a Chimeric Signaling Receptor (CSR) comprising i) a scFv that specifically binds GPC3, the scFv comprising a V comprising the amino acid sequence of SEQ ID NO:64_HDomain and V comprising the amino acid sequence of SEQ ID NO 65_LA domain; ii) a transmembrane domain, and iii) an immune cell costimulatory molecule fragment (fCSM) comprising an intracellular signaling domain capable of providing a costimulatory signal to an immune cell, wherein the costimulatory molecule fragment comprises the amino acid sequence of any one of SEQ ID NOs 51-56 and 86-89. In some embodiments, the CSR transmembrane domain is derived from CD 8. In some embodiments, the CSR comprises a transmembrane protein fragment (fTMP), wherein the transmembrane protein fragment comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the CSR comprises a spacer peptide following the ligand binding domain. In some embodiments, the spacer peptide comprises the amino acid sequence of SEQ ID NO 103 or 104. In some embodiments, the CSR domain is selected according to any one of the CSRs listed in table 3. In some embodiments, the CSR ligand binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 82.

Also contemplated are methods of treating a target antigen-associated disease in an individual in need thereof comprising administering to the individual a composition comprising a plurality of immune cells expressing different catrs and/or different CSRs. Thus, in some embodiments, the composition is a heterogeneous caccr plus CSR immune cell composition as described herein, according to any of the methods for treating a target antigen-associated disease in an individual as described herein.

In some embodiments, the subject is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. In some embodiments, the individual is less than about 60 years of age (including, e.g., less than any of about 50, 40, 30, 25, 20, 15, or 10 years of age). In some embodiments, the individual is older than about 60 years (including, e.g., older than any of about 70, 80, 90, or 100 years). In some embodiments, the individual is diagnosed with, or is environmentally or genetically predisposed to, one or more of the diseases or disorders described herein (such as cancer or a viral infection). In some embodiments, the individual has one or more risk factors associated with one or more of the diseases or disorders described herein.

In some embodiments, the caTCR plus CSR immune cell compositions of the invention are administered in combination with a second, third, or fourth agent (comprising, for example, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder involving expression of a target antigen. In some embodiments, the caTCR plus CSR immune cell composition is administered in combination with a cytokine (such as IL-2). In some embodiments, the caTCR plus CSR immune cell composition is administered in combination with an agent that increases expression of MHC proteins and/or enhances surface presentation of peptides by MHC proteins. In some embodiments, the agent comprises, for example, an IFN receptor agonist, an Hsp90 inhibitor, an enhancer of p53 expression, and a chemotherapeutic agent. In some embodiments, the agent is an IFN receptor agonist, comprising, for example, IFN γ, IFN β, and IFN α. In some embodiments, the agent is an Hsp90 inhibitor, comprising, for example, tanespimycins (17-AAG), apramycin (17-DMAG), restamycin (IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932(CUDC-305), PU-H71, Ganetespib (STA-9090), NVP-AUY922(VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL 888. In some embodiments, the agent is a p53 expression enhancer comprising, for example, 5-fluorouracil and Nutlin-3. In some embodiments, the agent is a chemotherapeutic agent, comprising, for example, topotecan (topotecan), etoposide (etoposide), cisplatin (cissplatin), paclitaxel (paclitaxel), and vinblastine (vinblastine).

In some embodiments, there is provided a method of treating a target antigen positive disease in an individual in need thereof, comprising administering to the individual a caTCR plus CSR immune cell composition according to any of the embodiments described herein in combination with a cytokine (such as IL-2). In some embodiments, the caTCR plus CSR immune cell composition is administered concurrently with the cytokine. In some embodiments, the caTCR plus CSR immune cell composition and cytokine are administered sequentially.

In some embodiments, a method of treating a target antigen positive disease in an individual in need thereof is provided, wherein cells expressing the target antigen do not normally present or present at a relatively low level on their surface a complex comprising the target antigen and an MHC class I protein, the method comprising administering to the individual a caccr plus CSR immune cell composition according to any one of the embodiments described herein in combination with an agent that increases expression of the MHC class I protein and/or enhances surface presentation of the MHC class I protein to the target antigen. In some embodiments, the agent comprises, for example, an IFN receptor agonist, an Hsp90 inhibitor, an enhancer of p53 expression, and a chemotherapeutic agent. In some embodiments, the agent is an IFN receptor agonist, comprising, for example, IFN γ, IFN β, and IFN α. In some embodiments, the agent is an Hsp90 inhibitor, comprising, for example, tanespimycins (17-AAG), apramycin (17-DMAG), restamycin (IPI-504), IPI-493, CNF2024/BIIB021, MPC-3100, Debio 0932(CUDC-305), PU-H71, Ganetespib (STA-9090), NVP-AUY922(VER-52269), HSP990, KW-2478, AT13387, SNX-5422, DS-2248, and XL 888. In some embodiments, the agent is a p53 expression enhancer comprising, for example, 5-fluorouracil and Nutlin-3. In some embodiments, the agent is a chemotherapeutic agent comprising, for example, topotecan, etoposide, cisplatin, paclitaxel, and vinblastine. In some embodiments, the caTCR plus CSR immune cell composition is administered concurrently with the agent. In some embodiments, the caTCR plus CSR immune cell composition and the agent are administered sequentially.

In some embodiments, there is provided a method of treating a target antigen-associated disease (such as cancer or a viral infection) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid encoding a caccr and CSR according to any of the embodiments described herein. Methods of gene delivery are known in the art. See, for example, U.S. patent nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety.

Cancer treatment can be assessed, for example, by tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Methods of determining the efficacy of a therapy may be used, including measuring the response, for example, via radiographic imaging.

In some embodiments, treatment efficacy is measured as percent tumor growth inhibition (TGI%), calculated using equation 100- (T/C x 100), where T is the average relative tumor volume of treated tumors and C is the average relative tumor volume of untreated tumors. In some embodiments, the% TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or more than 95%.

Treatment of viral infections can be assessed, for example, by viral load, duration of survival, quality of life, protein expression and/or activity.

Disease and disorder

In some embodiments, the caTCR plus CSR immune cells may be useful for treating cancer associated with a target antigen. Cancers that can be treated using any of the methods described herein include non-vascularized, or substantially non-vascularized, tumors, as well as vascularized tumors. The types of cancers to be treated with the calcr plus CSR immune cells of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies such as sarcomas, carcinomas, and melanomas.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and medulloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (refractory and advanced forms), multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, gastric carcinoma, lymphoid malignancies, pancreatic carcinoma, breast carcinoma, lung carcinoma, ovarian carcinoma, prostate carcinoma, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid carcinoma (e.g., medullary thyroid carcinoma and papillary thyroid carcinoma), pheochromocytoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, wilms' tumor, cervical carcinoma (e.g., cervical carcinoma and pre-invasive cervical dysplasia), colorectal carcinoma, anal canal, or anal carcinoma, Vaginal cancer, vulvar cancer (e.g., squamous cell carcinoma, intraepithelial cancer, adenocarcinoma, and fibrosarcoma), penile cancer, oropharyngeal cancer, esophageal cancer, head cancer (e.g., squamous cell carcinoma), neck cancer (e.g., squamous cell carcinoma), testicular cancer (e.g., sperm cell cancer, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, leydig cell tumor, fibroma, fibroadenoma, adenomatous tumors, and lipoma), bladder cancer, kidney cancer, melanoma, uterine cancer (e.g., endometrial carcinoma), urothelial cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer, and bladder cancer), and CNS tumors (such as gliomas (such as brain stem glioma and mixed gliomas), glioblastomas (also referred to as glioblastoma multiforme), astrocytoma, CNS lymphoma, and cervical carcinoma, Blastoma, neural tube blastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

Cancer treatment can be assessed, for example, by tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Methods of determining the efficacy of a therapy may be used, including measuring the response, for example, via radiographic imaging.

In other embodiments, the caTCR plus CSR immune cells may be suitable for treating infectious diseases by targeting pathogen-associated (such as virus-encoded) antigens. The infection to be prevented or treated may be caused, for example, by a virus, a bacterium, a protozoan or a parasite. The target antigen may be a pathogenic protein, polypeptide or peptide that causes a disease caused by the pathogen, or is capable of inducing an immune response in a host infected with the pathogen. Pathogenic antigens that can be targeted by the caTCR plus CSR immune cells include, but are not limited to, antigens derived from: acinetobacter baumannii (Acinetobacter baumannii), Bordetella (Anaplas magenis), phagocytophilic Anaplasma (Anaplama phaseophophilum), hookworm brasiliensis (Ancylostoma brachziliense), Ancylostoma duodenale (Ancylostomum), Cryptococcus haemolyticus (Arcanobacter haemolyticus), Ascaris hominis (Ascaris lumiracoides), Aspergillus (Aspergillus niger), Astroviridae (Astroviridae), Babesia (Babesia genus), Bacillus anthracis (Bacillus ankhracus), Bacillus cereus (Bacillus cereus), Kleinsis (Bartonella henselae), BK virus, human cysticercosis (Blastocystis hominis), Blastomyces dermatitis (Blastomyces), Bordetella (Bordetella pertussis strain), Borrelia (Borrelia), Borrelia cepacis brueckea, Borrelia (Borrelia), Borrelia cepacia and Borrelia (Borrelia), Borrelia (Borrelia species Borrelia), Borrelia (Borrelia abortus), Borrelia (Borrelia) and Borrelia (Borrelia species Borrelia (Borrelia), Borrelia (Borrelia) are strains, Burkholderia rhinoceros (Burkholderia mellea), Burkholderia pseudomallei), Caliciviridae (Caliciviridae family), Corynebacterium (Camphyllobacter genus), Candida albicans (Candida), Candida spp, Chlamydia trachomatis (Chlamydia trachorinata), Chlamydia pneumoniae (Chlamydia pneumoniae), Chlamydia psilon, Chlamydia psittaci (Chlamydophila psittaci), CJD prion, Clonorchis sinensis (Clonorchis sinensis), Clostridium botulinum (Clostridium bortutulinum), Clostridium difficile (Clostridium difficile), Clostridium perfringens (Clostridium perfringens), Clostridium difficinodosum (Clostridium difficile), Clostridium botulinum (Clostridium difficile), Clostridium difficile (Clostridium difficile), Clostridium difficile (Clostridium diffici, Cytomegalovirus (CMV), dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), crisp dicamba amoeba (Dientamoeba fragiliss), Ebola Virus (EBOV), Cytospora, Chaphieriensis (Ehrlichhachaffeensis), Ehrlichia Evosa (Ehrlichia ewingii), Ehrlichia (Ehrlichia genus), Entamoeba histolytica (Entamoeba histolytica), Enterococcus (Enterococcus genus), Enterovirus (Enterovirus) (mainly Saxach. A Virus and Enterovirus 71 (71)), Epidermophyton (Epidermophyton sp), Epstein-Barr Virus (Epstein-Barr Virus; EBV), Escherichia coli O157: h7, Escherichia coli O111 and Escherichia coli O104: h4, Fasciola hepatica and Hibiscus giganteus, FFI prions, filarial superfamily (Filarioideae superfamily), Flaviviviruses, Francisella farinosa (Francisella tularensis), Fusobacterium (Fusobacterium genus), Geotrichum candidum, Giardia lamblia (Giardia intestinalis), Gracilaria (Gathostomia spp), GSS prions, Guararito virus (Guianarito virus), Haemophilus duchensis (Haemophilus creyii), Haemophilus influenzae (Haemophilus fluuezae), Helicobacter pylori (Helicobacter pylori), Henry virus (Henry), hepatitis A, hepatitis B (HSV) and hepatitis C1-2), hepatitis C and hepatitis C2, Hepatitis C (HCV), hepatitis C1-2, hepatitis C and hepatitis C2), hepatitis C and hepatitis C2, hepatitis C and hepatitis C, Venerigeron wilsonii (Hortaea wereckii), human bocavirus (HBoV), human herpesvirus 6(HHV-6) and human herpesvirus 7(HHV-7), human interstitial pneumovirus (hMPV), Human Papilloma Virus (HPV), human parainfluenza virus (HPIV), human T-cell leukemia virus 1(HTLV-1), Japanese encephalitis virus, JC virus, junin virus (Junlvirus), Kaposi's Sarcoma-associated herpes virus (Kaposi's Sarcoma associated herpesvirus; KSHV), Kingella Kingensis kinase, Klebsiella granulosa (Klebsiella grandis), Kuru prion (Kuru prion), Lassa virus (Lassa virus), Legionella pneumophila (Legiophila), Listeria monocytogenes (Legiomonas meningitidis), Leginis meningitidis (Legiophaga), Legiophaga meningitis (Legiophaga), Lemongolitis virus (Legiophaga meningitis), Leginia meningitis (Legiophaga virus (Leginia), Leginia meningitis (Leginia), Leginia virus (Leginia), Leginia meningitis, Leginia virus (Leginia virus), Leginosa, Leginia virus (Leginia virus), Leginis, Leginosa, Leginia virus (Leginis, Leginia virus (Leginis, marcrobovirus (Machupo virus), Malassezia spp, Marburg virus (Marburg virus), measles virus, Metaplexis multocida (Metaplexis syaca), Microsporum (Microsporidia phylum), Molluscum Contagiosum Virus (MCV), mumps virus, Mycobacterium leprae and Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerosa, Mycoplasma pneumoniae, Trichosporoides (Naegleria foeri), Uncaria americana (Necator americanus), Diplococcus gonorrhoeae (Neissornorhorea), Diplococcus meningitidis (Neisseria meningitidis), Agrobacterium stellatum (Nocardia terrierella), Nocardia spp, human Pantoea (Neocallicarpa), Paralichia (Paralichia), Paralichia fasciata (Paralichia fasciata spp), Paralichia fasciata (Paralichia sp), Paralichia fasciata (Paralichia fasciata), Paralichia fasciata (Paralichia fasciata), Farformes), pasteurella (Pasteurella), Plasmodium (Plasmodium genus), Geigera (Pneumocystis jirovacii), poliomyelitis (Poliovirus), Rabies (Rabis virus), Respiratory Syncytial Virus (RSV), Rhinovirus (Rhinocavirus/Rhinovirus), Rickettsia (Rickettsia akari), Rickettsia (Rickettsia auricular), Rickettsia (Rickettsia) Rickettsia, Rickettsia (Rickettsia prokaryoti), Rickettsia (Rickettsia prowazekii), Rickettsia (Rickettsia orientalis), Rickettsia typhi (Rickettsia typhi), Dongfei non-Rickettsia valley fever virus, Rotavirus (Rotavirus), German virus (Ruzeberia virus), Salmonella (Sacubiella typhi), Salmonella (Salmonella typhimurium), Sargentamia (Schizosaccharomyces spp), Sarcophytrium (Sarcochinus), Sarcocystus (Sarcocystus), Sarcocystus (Rickensis virus), Sarcocystis (Rickensis virus (Ricken), Sarcocystis virus (Sarcocystis), Sarcocystis virus (Sar, Staphylococcus (Staphylococcus genus), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus pyogenes (Streptococcus pycnosis), Strongyloides stercoralis (Strongyloides stercoralis), Taenia (Taeniderived species), Toxobacter aquilegia (Taenidium solium), Tick-borne encephalitis virus (Tiborn encephalis virus; TBEV), ascaris canis (Toxocara canis) or ascaris felis (Toxocara catei), Toxoplasma gondii (Toxoplasma gondii), Treponema pallidum (Treponema pallidum), Trichinella spiralis (Trichinella spiris), Trichosta vaginalis (Trichosta vasica), Trichosta trichophytoides (Trichosta), Trichosporoides (Trichosporoides), Trichosta venes (Trichosta), Trichosta virus (Trichosporos), Trichosta virus (Vasella maculata), Vasella viridis (Vasella viridis), Trypus (Vazii), Trypus flavivirus), Trypus flavipes virus (Vazicola), Trypus), Tryporus virus (Vazianum virus (Vazianulus fascicularia), Trypus flavus), Trypus flavipes virus (Vazianum), Trypus flavivirus, Vazianulus fasciolius flavus serous (Vazianum), Trypus flavus serous, Vazianum virus (Vazianulus fasciatus (Vazi, Vibrio cholerae (Vibrio cholerae), West Nile virus (West Nile virus), Western equine encephalitis virus (Western infectious phalitis virus), Pennella pannicus (Wuchereria bancrofti), Yellow fever virus (Yellow fever virus), Yersinia enterocolitica (Yersinia enterocolitica), Yersinia pestis (Yersinia pestivirus), and Yersinia pseudotuberculosis (Yersinia pseudotuberculosis).

In some embodiments, the cadcr plus CSR immune cells are used to treat an oncogenic infectious disease, such as an infection caused by an oncogenic virus. Oncogenic viruses include, but are not limited to, CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1, HIV-1, and HCV. The target antigen of the caTCR may be a viral oncoprotein including, but not limited to, Tax, E7, E6/E7, E6, HBx, EBNA proteins (e.g., EBNA 3A, EBNA 3C and EBNA 2), v-cyclin, LANA1, LANA2, LMP-1, K-bZIP, RTA, KSHV K8, and fragments thereof. See Ahuja, Richa et al, curr. sci., 2014.

Article and kit

In some embodiments of the invention, an article of manufacture is provided that contains materials suitable for use in the treatment of a target antigen-positive disease, such as cancer (e.g., adrenocortical, bladder, breast, cervical, biliary, colorectal, esophageal, glioblastoma, glioma, hepatocellular, head and neck, renal, leukemia, lung, lymphoma, melanoma, mesothelioma, multiple myeloma, pancreatic, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian, prostate, sarcoma, gastric, uterine, or thyroid cancer) or a viral infection (e.g., an infection caused by CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1, HIV-1, or HCV). The article of manufacture may comprise a container and a label or pharmaceutical insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. Generally, the container contains a composition effective for treating the diseases or conditions described herein, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immune cell presenting the caTCR and CSR of the invention on its surface. The label or package insert indicates that the composition is for use in treating a particular condition. The label or package insert will further include instructions for administering the caccr plus CSR immune cell composition to a patient. Also encompassed are articles of manufacture and kits comprising the combination therapies described herein.

The package insert refers to instructions that contain information regarding the indications, use, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products that are typically included in a package of commercially available therapeutic products. In some embodiments, the package insert indicates that the composition is for use in treating a target antigen-positive cancer (such as adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular cancer, head and neck cancer, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, gastric cancer, uterine cancer, or thyroid cancer). In other embodiments, the package insert indicates that the composition is used to treat a target antigen positive viral infection (e.g., an infection caused by CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1, HIV-1, or HCV).

Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Also provided are kits suitable for use, optionally in combination with an article of manufacture, for various uses, such as for the treatment of a target antigen positive disease or disorder described herein. The kits of the invention comprise one or more containers comprising a caTCR plus CSR immune cell composition (or unit dosage form and/or article of manufacture), and in some embodiments further comprise another agent (such as an agent described herein) and/or instructions for use according to any of the methods described herein. The kit may further comprise instructions for selecting an individual suitable for treatment. The instructions provided in the kits of the invention are written instructions typically on a label or pharmaceutical instructions (e.g., a sheet of paper contained in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc) are also acceptable.

For example, in some embodiments, a kit comprises a composition comprising immune cells presenting catrs and CSRs on their surface. In some embodiments, the kit comprises: a) a composition comprising an immune cell that presents caTCR and CSR on its surface, and b) an effective amount of at least one additional agent, wherein the additional agent increases expression of an MHC protein and/or enhances surface presentation of a peptide by the MHC protein (e.g., an IFN γ, IFN β, IFN α, or Hsp90 inhibitor). In some embodiments, the kit comprises: a) a composition comprising immune cells that present catrs and CSRs on their surface, and b) instructions for administering the catrs plus CSR immune cell composition to a subject to treat a target antigen-positive disease (such as cancer or a viral infection). In some embodiments, the kit comprises: a) a composition comprising immune cells that present catrs and CSRs on their surface, b) an effective amount of at least one other agent, wherein the other agent increases expression of MHC proteins and/or enhances surface presentation of peptides by MHC proteins (e.g., an IFN γ, IFN β, IFN α, or Hsp90 inhibitor), and c) instructions for administering the catrs plus CSR immune cell composition and the other agent to a subject for treating a target antigen-positive disease, such as cancer or a viral infection. The caTCR plus CSR immune cell composition and other agents can be present in separate containers or in a single container. For example, a kit can include one unique composition or two or more compositions, where one composition includes the caTCR plus CSR immune cells and another composition includes other agents.

In some embodiments, the kit comprises: a) one or more compositions comprising a caTCR and a CSR, and b) instructions for combining the caTCR and the CSR with an immune cell (such as an immune cell derived from the subject, e.g., a T cell or a natural killer cell) to form a composition comprising an immune cell presenting the caTCR and the CSR on its surface and administering the caTCR plus CSR immune cell composition to the subject to treat a target antigen positive disease (such as cancer or a viral infection). In some embodiments, the kit comprises: a) one or more compositions comprising a caTCR and CSR, and b) an immune cell (such as a cytotoxic cell). In some embodiments, the kit comprises: a) one or more compositions comprising a caTCR and a CSR, b) an immune cell (such as a cytotoxic cell), and c) instructions for combining the caTCR and the CSR with the immune cell to form a composition comprising an immune cell that presents the caTCR and the CSR on its surface and administering the caTCR plus CSR immune cell composition to a subject to treat a target antigen positive disease (such as cancer or a viral infection).

In some embodiments, the kit comprises a nucleic acid (or a set of nucleic acids) encoding a caTCR and a CSR. In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding the caTCR and CSR, and b) a host cell (such as an immune cell) that expresses the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding a caTCR and a CSR, and b) instructions for: i) expressing the caTCR and CSR in a host cell (such as an immune cell, e.g., a T cell), ii) preparing a composition comprising the host cell expressing the caTCR and CSR, and iii) administering the composition comprising the host cell expressing the caTCR and CSR to a subject to treat a target antigen-positive disease (such as cancer or a viral infection). In some embodiments, the host cell is derived from an individual. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding a caTCR and a CSR, b) a host cell (such as an immune cell) that expresses the nucleic acid (or the set of nucleic acids), and c) instructions for i) expressing the caTCR and the CSR in the host cell, ii) preparing a composition comprising the host cell that expresses the caTCR and the CSR, and iii) administering the composition comprising the host cell that expresses the caTCR and the CSR to the subject to treat a target antigen-positive disease (such as cancer or a viral infection).

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kit may optionally provide additional components such as buffers and instructional information. The present application thus also provides articles of manufacture comprising vials (such as sealed vials), bottles, jars, flexible packages, and the like.

The instructions relating to the use of the caTCR plus CSR immune cell composition generally include information regarding the dosage, time course of administration, and route of administration of the desired treatment. The container may be a unit dose, a bulk (e.g., multi-dose pack), or a sub-unit dose. For example, a kit can be provided that contains a sufficient dose of a caccr plus CSR immune cell composition as disclosed herein to provide effective treatment to an individual for an extended period of time, such as any of one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. The kit may also comprise a plurality of unit doses of the caTCR and CSR, and pharmaceutical compositions and instructions for use and packaged in sufficient quantities for storage and use in pharmacies, such as hospital pharmacies and pharmacy.

Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Exemplary embodiments