Engineered TCR complexes and methods of using same

Abstract

Engineered T cell receptor (TCR) complexes and methods of using same are provided. Thus, a TCR is provided, comprising a TCR alpha polypeptide and a TCR beta polypeptide, the TCR alpha and beta polypeptides lacking a binding domain, and the TCR alpha and beta polypeptides comprising amino acid modifications that allow for presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR. Polynucleotides encoding the TCRs, T cells expressing the TCR complexes, and methods of using same are also provided.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to European Patent Application No. 21189516.4, filed August 3, 2021, and U.S. Provisional Patent Application No. 63 / 345,966, filed May 26, 2022, the contents of which are incorporated by reference in their entireties herein.

[0002] Description of sequence listing A file entitled 93251.xml, created on July 21, 2022, containing 274,432 bytes, and submitted contemporaneously with the filing of this application, is hereby incorporated by reference.

[0003] The present invention, in some embodiments, relates to engineered TCR complexes and methods of using same.

[0004] Cancer immunotherapy, including cell-based, antibody, and cytokine therapies, has emerged in recent years as a promising strategy for treating various types of cancer due to its potential to circumvent genetic and cellular mechanisms of drug resistance and target tumor cells while sparing healthy tissue.

[0005] For example, cell-based therapy using T cells with T cell receptors (TCRs) specific for antigens differentially expressed in association with MHC class I molecules on cancer cells or with chimeric antigen receptors (CARs) containing an antigen recognition portion (e.g., single-chain variable fragments (scFvs)) and a T cell activation portion has been shown to exert antitumor effects in some types of cancer, such as hematological malignancies. However, TCRs are limited in their recognition spectrum and MHC class, and in addition, when introducing exogenous TCRs into T cells, there is a risk of hybridization between exogenous and endogenous chains, which may induce recognition of self-antigens [Van Loenen MM et al., Proc Natl Acad Sci USA. 2010; 107: 10972-7]. On the other hand, despite their advantages, CART cells have significant deficiencies that need to be resolved to allow full utilization of the technology in clinical treatment, such as, for example, aggressive proliferation in the presence of heavy tumor burden resulting in severe side effects, tumor lysis syndrome and cytokine release syndrome, and the generation of tumor escape mutants that have lost the target antigen during treatment [Morgan RA et al. (2010) Mol Ther. 18: 843-51; Brudno JN et al. (2016); Blood. American Society of Hematology; pp. 3321-30; and Grupp SA et al. (2013) N Engl J Med 368: 1509-18]. Moreover, the use of allogeneic T cells for treatment poses the risk of adverse recognition of self-antigens resulting in graft-versus-host disease (GVHD).

[0006] Antibody-based cancer immunotherapies, such as monoclonal antibodies, antibody fusion proteins, and antibody drug conjugates (ADCs), rely on the recognition of cell surface molecules differentially expressed on cancer cells compared to noncancerous cells and / or immune checkpoint blockade. Binding of antibody-based immunotherapies to cancer cells can result in cancer cell death through a variety of mechanisms, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct cytotoxic activity of the payload from the antibody-drug conjugate (ADC), or inhibitory checkpoint blockade. Many of these mechanisms are initiated through binding of the Fc domain of the cell-binding antibody to specialized cell surface receptors (Fc receptors) on hematopoietic cells. Recently, another type of antibody-based therapy has been proposed, namely anti-CD3 bispecific antibodies, such as mosunetuzumab, odronextamab, and blinatumomab. These bispecific antibodies bind CD3-positive T cells to tumor-associated antigens, thus promoting the T cells to attack tumor cells.

[0007] Immunotherapies combining principles of antibody-based therapy, CAR T cell and / or TCR-based immunotherapy have been disclosed [see, e.g., Choi BD et al. (2019) Nature Publishing Group; 37:1049-58; Chandran and Klebanoff (2019) Immunological Reviews. 290:127-147; Benjamin R. (2020) Lancet 396:1885-94; Liu et al. (2021) Sci. Transl. Med. 13, eabb5191; Helsen CW et al. (2018) Nature Communications 9:3049; Baeuerle PA et al. (2019) Nature Communications 10:2087].

[0008] Additional background art can be found in International Patent Application Publication Nos. WO2019222275, WO2021035170, and WO2015143224; U.S. Patent Application Publication Nos. US20190388472 and US20190070248; Japanese Patent No. 2017514471; and Including Russian Patent No. RU2725542. Summary of the Invention

[0009] According to one aspect of some embodiments of the invention, there is provided a T cell receptor (TCR) complex comprising a TCR alpha polypeptide and a TCR beta polypeptide, wherein the TCR lacks an antigen-binding domain and the CD3 polypeptide lacks a heterologous antigen-binding domain, and wherein the TCR complex is capable of being presented on the surface of a T cell expressing the TCR.

[0010] According to one aspect of some embodiments of the present invention, there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, wherein the TCR lacks an antigen-binding domain, and the TCR alpha and beta polypeptides comprise amino acid modifications that enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR.

[0011] According to some embodiments of the invention, the TCR comprises a heterodimerisation moiety.

[0012] According to some embodiments of the invention, the heterodimerization moiety comprises a cysteine ​​in each of the TCR α and TCR β polypeptides.

[0013] According to some embodiments of the invention, the TCR alpha and TCR beta polypeptides comprise human TCR alpha and human TCR beta polypeptides.

[0014] According to some embodiments of the invention, the TCR alpha and TCR beta polypeptides include chimeric human and mouse TCR alpha and chimeric human and mouse TCR beta polypeptides.

[0015] According to one aspect of some embodiments of the invention there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, the TCR lacking an antigen binding domain and a heterologous extracellular binding domain, the TCR alpha and beta polypeptides comprising amino acid modifications which enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR, the modifications comprising (i) comprising a T48C amino acid substitution corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and a S57C amino acid substitution corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12, and / or (ii) TCR alpha and TCR beta polypeptides include chimeric human and mouse TCR alpha and chimeric human and mouse TCR beta polypeptides.

[0016] According to one aspect of some embodiments of the invention there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, wherein the TCR lacks an antigen binding domain, and the TCR alpha and beta polypeptides comprise amino acid modifications which enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR, the modifications comprising: (i) the T48C, S116L, G119V, and F120L amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and the S57C mutation corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12, and / or (ii) P91S, E92D, S93V, and S94P amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO:9, and E18K, S22A, F133I, E / V136A, and Q139H amino acid substitutions corresponding to the human TCR beta polypeptide set forth in SEQ ID NO:12.

[0017] According to some embodiments of the invention, the (ii) modification further comprises S116L, G119V, and F120L amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO:9.

[0018] According to some embodiments of the invention, the modification further comprises S116L, G119V, and F120L amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO:9.

[0019] According to some embodiments of the invention, the (ii) modification includes P91S, E92D, S93V, and S94P amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and E18K, S22A, F133I, E / V136A, and Q139H amino acid substitutions corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12.

[0020] According to some embodiments of the invention, the TCR alpha polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20-23, 47, 111, 114, and 117-120.

[0021] According to some embodiments of the invention, the TCR α polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20-23, 47, 111, and 117-120.

[0022] According to some embodiments of the invention, the TCR α polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 17, and 20-23.

[0023] According to some embodiments of the invention, the TCR α polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, and 20-23.

[0024] According to some embodiments of the invention, the TCR α polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 20-23.

[0025] According to some embodiments of the present invention, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122.

[0026] According to some embodiments of the present invention, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-11, 15-16, 18-19, and 24-25.

[0027] According to some embodiments of the present invention, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, and 24-25.

[0028] According to an aspect of some embodiments of the present invention there is provided at least one polynucleotide encoding a TCR.

[0029] According to an aspect of some embodiments of the present invention there is provided a transduced cell expressing a TCR or at least one polynucleotide.

[0030] According to an aspect of some embodiments of the invention there is provided a transduced T cell expressing a TCR complex or a TCR or at least one polynucleotide.

[0031] According to an aspect of some embodiments of the invention there is provided a method of producing a TCR-expressing cell, the method comprising introducing at least one polynucleotide into a cell under conditions allowing expression of the TCR.

[0032] According to an aspect of some embodiments of the invention there is provided a method of expressing a TCR in a T cell, the method comprising introducing at least one polynucleotide into the T cell under conditions allowing expression of the TCR.

[0033] According to some embodiments of the invention, the introducing is effected in vitro or ex vivo.

[0034] According to some embodiments of the invention, the cells do not express an endogenous TCR.

[0035] According to some embodiments of the invention, the T cells do not express an endogenous TCR.

[0036] According to some embodiments of the invention, the method further comprises downregulating expression of an endogenous TCR.

[0037] According to some embodiments of the invention, the method further comprises downregulating expression of an endogenous TCR prior to the introducing.

[0038] According to an aspect of some embodiments of the invention there is provided a method of treating a disease associated with a pathological cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell and a therapeutic composition capable of binding to the pathological cell and the TCR complex, thereby treating the disease in the subject.

[0039] According to an aspect of some embodiments of the invention there is provided a T cell and a therapeutic composition capable of binding to a pathological cell and a TCR complex for use in treating a disease associated with a pathological cell in a subject in need thereof.

[0040] According to an aspect of some embodiments of the invention there is provided a method of treating a disease associated with a pathological cell in a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of a T cell and a therapeutic composition capable of binding to a TCR complex comprising a pathological cell and a TCR, thereby treating the disease in the subject.

[0041] According to an aspect of some embodiments of the invention there is provided a therapeutic composition capable of binding to a T cell and a TCR complex comprising a pathological cell and a TCR for use in treating a disease associated with a pathological cell in a subject in need thereof.

[0042] According to some embodiments of the invention, the T cells are allogeneic to the subject.

[0043] According to one aspect of some embodiments of the invention, an article of manufacture is provided that includes packaging material for packaging a cell and a therapeutic composition capable of binding to a TCR complex comprising a pathological cell and a TCR.

[0044] According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising packaging material for packaging a T cell and a therapeutic composition capable of binding to a pathological cell and a TCR complex.

[0045] According to some embodiments of the invention, the therapeutic composition comprises an anti-CD3 antibody.

[0046] Non-limiting examples of anti-CD3 antibodies that can be used in certain embodiments of the invention include L2K, TR66, OKT3, SP34, UCHT1, F6A, humanized UCHT1, SK7, and HIT3A.

[0047] According to some embodiments of the invention, the anti-CD3 antibody is selected from the group consisting of L2K, TR66, and OKT3.

[0048] According to some embodiments of the invention, the pathological cells express CD19 and the therapeutic composition comprises blinatumomab.

[0049] According to some embodiments of the invention, the pathological cells express EpCAM and the therapeutic composition comprises MT110.

[0050] According to some embodiments of the invention, the pathological cell is a cancer cell.

[0051] According to some embodiments of the invention, the cancer is selected from the group consisting of lymphoma, leukemia, glioblastoma, colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, and skin cancer.

[0052] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. [Brief description of the drawings]

[0053] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Referring now specifically to the drawings in detail, it is emphasized that the details shown are by way of example and for purposes of illustrative discussion of embodiments of the present invention. In this regard, the description with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0054] The drawings are as follows: [Figure 1A] Schematic diagrams of engineered TCR complexes of some embodiments of the present invention are shown. Figure 1A demonstrates a TCR complex comprising TCR α and β chains lacking the variable α and β regions. This TCR is referred to herein as "blunt truncated TCR (BluT)". Figure 1B demonstrates a TCR complex comprising mismatched pairing of exogenous TCR α and β chains lacking the variable regions with endogenous TCR α and β chains. [Figure 1B] Schematic diagrams of engineered TCR complexes of some embodiments of the present invention are shown. Figure 1A demonstrates a TCR complex comprising TCR α and β chains lacking the variable α and β regions. This TCR is referred to herein as "blunt truncated TCR (BluT)". Figure 1B demonstrates a TCR complex comprising mismatched pairing of exogenous TCR α and β chains lacking the variable regions with endogenous TCR α and β chains. [Figure 2A-1]Schematic diagram of engineered TCRs of some embodiments of the invention. Where indicated, three mutations were introduced in the transmembrane domain of the α chain: S116L, G119V, and F120L (designated LVL). Where indicated, cysteines were introduced in both the α and β chains (designated CYS): T48C in the α chain and S57C in the β chain. Where indicated, several mutations were introduced in the extracellular domains of the α and β chains as follows: α chain mutations P91S, E92D, S93V, S94P; β chain mutations: E18K, S22A, F133I, E / V136A, Q139H (designated mm). Where indicated, cysteines were introduced into both the α and β chains: CL12 in the α chain and S17C in the β chain, Y43C in the α chain and L63C in the β chain, S61C in the α chain and R79C in the β chain, L12C in the α chain and F14C in the β chain, V22C in the α chain and F14C in the β chain, Y10C in the α chain and S17C in the β chain, T45C in the α chain and D59C in the β chain, L50C in the α chain and S57C in the β chain, S61C in the α chain and S57C in the β chain, T45C in the α chain and S77C in the β chain, S15C in the α chain and V13C in the β chain, or S15C in the α chain and E15C in the β chain. Where indicated, some mutations were introduced into the α and β chains as follows: α-chain mutations S21F, T32I, A72T and β-chain mutations E18K, H23R, D39P, S54D (denoted Des). The F5P2A-Furin-V5 tag sequence was combined with P2A. [Figure 2A-2]Schematic diagram of engineered TCRs of some embodiments of the invention. Where indicated, three mutations were introduced in the transmembrane domain of the α chain: S116L, G119V, and F120L (designated LVL). Where indicated, cysteines were introduced in both the α and β chains (designated CYS): T48C in the α chain and S57C in the β chain. Where indicated, several mutations were introduced in the extracellular domains of the α and β chains as follows: α chain mutations P91S, E92D, S93V, S94P; β chain mutations: E18K, S22A, F133I, E / V136A, Q139H (designated mm). Where indicated, cysteines were introduced into both the α and β chains: CL12 in the α chain and S17C in the β chain, Y43C in the α chain and L63C in the β chain, S61C in the α chain and R79C in the β chain, L12C in the α chain and F14C in the β chain, V22C in the α chain and F14C in the β chain, Y10C in the α chain and S17C in the β chain, T45C in the α chain and D59C in the β chain, L50C in the α chain and S57C in the β chain, S61C in the α chain and S57C in the β chain, T45C in the α chain and S77C in the β chain, S15C in the α chain and V13C in the β chain, or S15C in the α chain and E15C in the β chain. Where indicated, some mutations were introduced into the α and β chains as follows: α-chain mutations S21F, T32I, A72T and β-chain mutations E18K, H23R, D39P, S54D (denoted Des). The F5P2A-Furin-V5 tag sequence was combined with P2A. [Figure 2B-1]Schematic diagram of engineered TCRs of some embodiments of the invention. Where indicated, three mutations were introduced in the transmembrane domain of the α chain: S116L, G119V, and F120L (designated LVL). Where indicated, cysteines were introduced in both the α and β chains (designated CYS): T48C in the α chain and S57C in the β chain. Where indicated, several mutations were introduced in the extracellular domains of the α and β chains as follows: α chain mutations P91S, E92D, S93V, S94P; β chain mutations: E18K, S22A, F133I, E / V136A, Q139H (designated mm). Where indicated, cysteines were introduced into both the α and β chains: CL12 in the α chain and S17C in the β chain, Y43C in the α chain and L63C in the β chain, S61C in the α chain and R79C in the β chain, L12C in the α chain and F14C in the β chain, V22C in the α chain and F14C in the β chain, Y10C in the α chain and S17C in the β chain, T45C in the α chain and D59C in the β chain, L50C in the α chain and S57C in the β chain, S61C in the α chain and S57C in the β chain, T45C in the α chain and S77C in the β chain, S15C in the α chain and V13C in the β chain, or S15C in the α chain and E15C in the β chain. Where indicated, some mutations were introduced into the α and β chains as follows: α-chain mutations S21F, T32I, A72T and β-chain mutations E18K, H23R, D39P, S54D (denoted Des). The F5P2A-Furin-V5 tag sequence was combined with P2A. [Figure 2B-2]Schematic diagram of engineered TCRs of some embodiments of the invention. Where indicated, three mutations were introduced in the transmembrane domain of the α chain: S116L, G119V, and F120L (designated LVL). Where indicated, cysteines were introduced in both the α and β chains (designated CYS): T48C in the α chain and S57C in the β chain. Where indicated, several mutations were introduced in the extracellular domains of the α and β chains as follows: α chain mutations P91S, E92D, S93V, S94P; β chain mutations: E18K, S22A, F133I, E / V136A, Q139H (designated mm). Where indicated, cysteines were introduced into both the α and β chains: CL12 in the α chain and S17C in the β chain, Y43C in the α chain and L63C in the β chain, S61C in the α chain and R79C in the β chain, L12C in the α chain and F14C in the β chain, V22C in the α chain and F14C in the β chain, Y10C in the α chain and S17C in the β chain, T45C in the α chain and D59C in the β chain, L50C in the α chain and S57C in the β chain, S61C in the α chain and S57C in the β chain, T45C in the α chain and S77C in the β chain, S15C in the α chain and V13C in the β chain, or S15C in the α chain and E15C in the β chain. Where indicated, some mutations were introduced into the α and β chains as follows: α-chain mutations S21F, T32I, A72T and β-chain mutations E18K, H23R, D39P, S54D (denoted Des). The F5P2A-Furin-V5 tag sequence was combined with P2A. [Figure 3A-1]We demonstrate the re-expression of CD3 in endogenous TCR negative T cells after expression of BluT. Figure 3A shows the generation of CD3- / TCR- T cells by electroporation with Cas9 RNP and gRNA targeting the TCR alpha chain (SEQ ID NO: 1), followed by magnetic bead purification of the CD3- / TCR- T cells. Figure 3B shows the re-expression of CD3 in the T cells shown in Figure 3A after infection with a construct encoding a truncated alpha chain containing an additional cysteine ​​and several transmembrane hydrophobic mutations and a truncated beta chain containing an additional cysteine, TRAC(Cys,LVL)-P2A-TRBC1(Cys), SEQ ID NO: 2, compared to no expression after infection with a construct encoding only the truncated alpha chain (TRAC, SEQ ID NO: 3), as determined by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 3A-2] We demonstrate the re-expression of CD3 in endogenous TCR negative T cells after expression of BluT. Figure 3A shows the generation of CD3- / TCR- T cells by electroporation with Cas9 RNP and gRNA targeting the TCR alpha chain (SEQ ID NO: 1), followed by magnetic bead purification of the CD3- / TCR- T cells. Figure 3B shows the re-expression of CD3 in the T cells shown in Figure 3A after infection with a construct encoding a truncated alpha chain containing an additional cysteine ​​and several transmembrane hydrophobic mutations and a truncated beta chain containing an additional cysteine, TRAC(Cys,LVL)-P2A-TRBC1(Cys), SEQ ID NO: 2, compared to no expression after infection with a construct encoding only the truncated alpha chain (TRAC, SEQ ID NO: 3), as determined by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 3B]We demonstrate the re-expression of CD3 in endogenous TCR negative T cells after expression of BluT. Figure 3A shows the generation of CD3- / TCR- T cells by electroporation with Cas9 RNP and gRNA targeting the TCR alpha chain (SEQ ID NO: 1), followed by magnetic bead purification of the CD3- / TCR- T cells. Figure 3B shows the re-expression of CD3 in the T cells shown in Figure 3A after infection with a construct encoding a truncated alpha chain containing an additional cysteine ​​and several transmembrane hydrophobic mutations and a truncated beta chain containing an additional cysteine, TRAC(Cys,LVL)-P2A-TRBC1(Cys), SEQ ID NO: 2, compared to no expression after infection with a construct encoding only the truncated alpha chain (TRAC, SEQ ID NO: 3), as determined by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 4-1] We demonstrate re-expression of CD3 in endogenous TCR-negative T cells following expression of BluT containing at least the T48C mutation in the α chain and the S57C mutation in the β chain. Endogenous TCR-negative T cells shown in Figure 3A were infected with the indicated constructs (see Figure 2B for a detailed description of each construct) and assessed by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 4-2] We demonstrate re-expression of CD3 in endogenous TCR-negative T cells following expression of BluT containing at least the T48C mutation in the α chain and the S57C mutation in the β chain. Endogenous TCR-negative T cells shown in Figure 3A were infected with the indicated constructs (see Figure 2B for a detailed description of each construct) and assessed by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Diagram 5]We demonstrate the re-expression of CD3 in endogenous TCR-negative T cells generated by targeting endogenous β or α and β chains after expression of BluT containing the T48C and LVL mutations in the α chain and the S57C mutation in the β chain. CD3- / TC-RT cells were generated by electroporation with Cas9 RNP and gRNA targeting the TCR β chain (SEQ ID NO: 45), or both the TCR α and β chains (SEQ ID NOs: 1 and 45), followed by magnetic bead purification of CD3- / TCR-T cells. Cells were then infected with the indicated constructs (see FIG. 2B for a detailed description of each construct) and assessed by flow cytometry using an anti-CD3 OKT3 antibody. EGFP serves as a marker of infection. [Figure 6-1] We demonstrate that expression of BluT containing mutations resulting in additional disulfide bonds between the α and β chains other than T48C in the α chain and S57C in the β chain does not allow re-expression of CD3. Endogenous TCR negative T cells shown in Figure 3A were infected with the indicated constructs (see Figure 2B for detailed description of each construct) and assessed by flow cytometry using anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 6-2] We demonstrate that expression of BluT containing mutations resulting in additional disulfide bonds between the α and β chains other than T48C in the α chain and S57C in the β chain does not allow re-expression of CD3. Endogenous TCR negative T cells shown in Figure 3A were infected with the indicated constructs (see Figure 2B for detailed description of each construct) and assessed by flow cytometry using anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 7] We demonstrate re-expression of CD3 in endogenous TCR-negative T cells following expression of BluT containing minimal mouse amino acid substitutions. Endogenous TCR-negative T cells shown in Figure 3A were infected with the indicated constructs (see Figure 2B for detailed description of each construct) and assessed by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 8A]We demonstrate the effect of deleting 6-21 amino acids from the N-terminus of the α-chain of BluT on re-expression of CD3 in endogenous TCR-negative T cells. Endogenous TCR-negative T cells shown in Figure 3A were infected with the constructs indicated (see Figure 2B for detailed description of each construct) and assessed by flow cytometry using an anti-CD3OKT3 antibody. EGFP serves as a marker of infection. [Figure 8B] Figure 3 shows the absence of CD3 expression in endogenous TCR-negative T cells after expression of the construct disclosed by International Patent Application Publication No. WO2020138371. Endogenous TCR-negative T cells shown in Figure 3A were infected with Ba9 (SEQ ID NO: 83 / 84) or α6 (SEQ ID NO: 81 / 82) and assessed by flow cytometry using anti-CD3 OKT3 antibody. EGFP serves as a marker of infection. [Figure 9] We demonstrate that T cells expressing BluT in combination with bispecific T cell engagers induce effective in vitro tumor cell lysis. Endogenous TCR-negative T cells shown in Figure 3A were infected with BluT containing T48C and LVL mutations, and a truncated β-chain containing S57C mutation (SEQ ID NO: 125 / 126) was incubated with Raji-F.Luc CD19+ lymphoma cells at the indicated E:T ratios in the presence or absence of blinatumomab (CD19 BiTE), and cytotoxicity was determined by measuring firefly luciferase activity. CD3-negative anti-CD19 CAR-T cells (SEQ ID NO: 127 / 128) and unmodified CD3-positive TCR-positive T cells were used as positive controls. Shown is the percentage of relative lysis calculated by dividing the test group RLU (relative light units) to the untreated tumor group RLU alone. Results are shown in (%). ****p<0.0001, ***p<0.001 by two-way ANOVA [Figure 10]We demonstrate that T cells expressing BluT in combination with bispecific T cell engagers induce effective in vivo anti-cancer effects. Endogenous TCR-negative T cells shown in Figure 3A were infected with BluT containing T48C and LVL mutations, and a truncated β-chain containing S57C mutation (SEQ ID NO: 125 / 126). Mice were then transplanted with CD19+Raji cells in combination with BluT-transduced T cells and treated with anti-CD19 BiTE blinatumomab where indicated. CD3-negative anti-CD19 CAR-T cells (SEQ ID NO: 127 / 128) and unmodified CD3-positive TCR-positive T cells were used as positive controls. Saline treatment served as a negative control. Mouse survival is shown as Kaplan-Meier curves. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] The present invention relates in some embodiments to engineered TCRs and methods of using same.

[0056] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0057] Cancer immunotherapy has emerged in recent years as a promising strategy for treating various types of cancer. For example, cell-based therapy using T cells with engineered T cell receptors (TCRs) or CART cells, antibody-based cancer immunotherapy, and combinations thereof have been shown to exert antitumor effects in several types of cancer.

[0058]

[0013] Referring now to specific embodiments of the invention, the inventors have designed and expressed a truncated TCR lacking the variable regions of the TCR α and β chains that are presented as part of the TCR complex on the surface of T cells lacking an endogenous TCR (Example 1 in the Examples section below). Moreover, T cells expressing the truncated TCR had anti-tumor effects both in vitro and in vivo, but only in the presence of the TCR and anti-CD3 mediator (Examples 2-3 in the Examples section below).

[0059] As a result, certain embodiments of the present teachings suggest T cells genetically engineered to express a truncated TCR in combination with a therapeutic composition capable of binding to the pathological cells on the one hand and to a TCR complex (e.g., a T cell engager antibody) on the other hand to treat diseases associated with the pathological cells (e.g., cancer).

[0060] Thus, according to one aspect of the invention there is provided a T cell receptor (TCR) complex comprising a TCR alpha polypeptide and a TCR beta polypeptide, wherein the TCR lacks an antigen-binding domain and the CD3 polypeptide lacks a heterologous antigen-binding domain, and wherein the TCR complex is capable of being presented on the surface of a T cell expressing the TCR.

[0061] According to further or alternative aspects of the present invention, there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, the TCR lacking an antigen-binding domain, and the TCR alpha and beta polypeptides comprising amino acid modifications that enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR.

[0062] According to further or alternative aspects of the invention there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, the TCR lacking an antigen binding domain and a heterologous extracellular binding domain, the TCR alpha and beta polypeptides comprising amino acid modifications which enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR, the modifications comprising (i) comprising a T48C amino acid substitution corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and a S57C amino acid substitution corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12, and / or (ii) The TCR α polypeptide and the TCR β polypeptide include chimeric human and mouse TCR α polypeptides and chimeric human and mouse TCR β polypeptides.

[0063] According to a further or alternative aspect of the invention there is provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR beta polypeptide, the TCR lacking an antigen binding domain, the TCR alpha and beta polypeptides comprising amino acid modifications which enable presentation of the TCR as a TCR complex on the surface of a T cell expressing the TCR, the modifications comprising (i) the T48C, S116L, G119V, and F120L amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and the S57C amino acid substitution corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12, and / or (ii) A TCR is provided, comprising P91S, E92D, S93V, and S94P amino acid substitutions corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO:9, and E18K, S22A, F133I, E / V136A, and Q139H amino acid substitutions corresponding to the human TCR beta polypeptide set forth in SEQ ID NO:12.

[0064] As used herein, the term "T cell receptor (TCR)" refers to a receptor comprising a heterodimer of a TCR alpha and a TCR beta polypeptide, and which can be presented (or is capable of being presented) as a TCR complex on the surface of a T cell that expresses the TCR.

[0065] As used herein, the term "TCR complex" refers to the complex formed by the association of CD3 with the TCR.

[0066] As used herein, the term "CD3" refers to the polypeptide expression products of the CD3G, CD3D, CD3E, or CD247 genes (gene IDs 917, 915, 916, 919, respectively), including CD3γ, CD3δ, CD3ε, and CD3zeta. According to certain embodiments, the CD3 is human CD3.

[0067] According to certain embodiments, CD3 is endogenous to the cell in which it is expressed.

[0068] According to certain embodiments, CD3 refers to the human CD3 gamma polypeptide as provided in the following Accession No. NP_000064 or SEQ ID NO:4.

[0069] According to certain embodiments, CD3 refers to human CD3δ as provided in the following accession numbers NP_000723, NP_001035741 or SEQ ID NO:5.

[0070] According to a particular embodiment, CD3 refers to human CD3ε as provided in the following accession number NP_000724 or SEQ ID NO:6.

[0071] According to certain embodiments, CD3 zeta (CD247) refers to the human as provided in the following accession numbers NP_000725, NP_932170, NP_001365444, NP_001365445, or SEQ ID NO:7.

[0072] As used herein, "CD3 polypeptide" or "CD3 chain" refers to full-length CD3 or a fragment or homolog thereof, which includes a signaling domain and maintains at least the ability of CD3 to be presented as a TCR complex on the surface of a CD3-expressing T cell. Such a homolog can be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or homologous to the polypeptides set forth in SEQ ID NOs: 4-7.

[0073] Thus, for example, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR alpha polypeptide, and a TCR beta polypeptide.

[0074] Methods for determining the presentation of TCR complexes are well known in the art and include, for example, flow cytometry or immunostaining using anti-CD3 or anti-TCRβ antibodies.

[0075] The TCRs disclosed herein lack an antigen-binding domain.

[0076] According to certain embodiments, the CD3 polypeptide lacks a heterologous antigen-binding domain.

[0077] According to certain embodiments in which the TCR comprises human TCR alpha and human TCR beta polypeptides that contain amino acid modifications that enable presentation of the TCR as a TCR complex on the surface of a T cell that expresses it, the CD3 polypeptide may comprise a heterologous antigen-binding domain.

[0078] As used herein, the term "heterologous" refers to an amino acid sequence or residues that are not native to the recited amino acid sequence (e.g., a TCR alpha polypeptide, a TCR beta polypeptide, a CD3 polypeptide) or that are not present entirely in the native sequence of the recited amino acid sequence, at least in localization.

[0079] Thus, according to certain embodiments, all components of the TCR complex lack an antigen-binding domain.

[0080] As used herein, the phrase "antigen-binding domain" refers to the binding of a TCR or antibody to an antigen in a specific manner, i.e., -4 It refers to a domain that contains an amino acid sequence that confers a Kd of less than or equal to M. Typically, such domains in the context of the present invention include the variable domains of the TCR α and β chains of a TCR, or the variable domains of the heavy and light chains of an antibody.

[0081] Thus, the TCR has a truncated naturally occurring antigen-binding domain, i.e. it lacks the naturally occurring antigen-binding domain of a naturally occurring TCR.

[0082] In other words, the TCR α and β polypeptides, as well as the CD3 polypeptide, of some embodiments of the present invention are not translationally fused to the antigen binding domain(s).

[0083] The TCR alpha and beta polypeptides or CD3 polypeptides of some embodiments may be joined to the antigen binding domain rather than as a translational fusion (e.g., an antibody comprising an antigen binding domain with binding specificity for a TCR complex).

[0084] According to other specific embodiments, the TCR alpha and / or beta polypeptides are not linked to a non-translationally fused antigen-binding domain.

[0085] According to certain embodiments, the TCR and / or CD3 polypeptides lack a binding domain capable of binding to a target displayed on the cell surface of, for example, a T cell target cell, i.e., the TCR alpha and beta polypeptides and / or the CD3 polypeptide are not translationally fused to a heterologous extracellular binding domain capable of binding to a target.

[0086] As used herein, the phrase "an extracellular binding domain capable of binding to a target" refers to a domain that has a binding affinity for a target (or binding partner) of interest, such as a target displayed on a target cell, i.e., -4 It refers to a proteinaceous moiety having a Kd equal to or less than M. Non-limiting examples of binding domains include receptor binding domains, ligand binding domains, hormone (e.g., leptin) binding domains, tags, and antigen binding domains, as further described hereinabove.

[0087] According to certain embodiments, the TCR lacks an antigen binding domain and a heterologous extracellular binding domain.

[0088] In other words, according to particular embodiments, the extracellular domain of the TCR polypeptide consists of the constant domains of the TCR α and β chains or a fragment or homologue thereof, or the constant domains and the hinge domain or a fragment or homologue thereof.

[0089] Assays for testing binding are well known in the art and include, but are not limited to, flow cytometry, immunostaining, Biolayer Interferometry Blitz® Assay, HPLC, surface plasmon resonance (e.g., Biacore).

[0090] As used herein, "TCR alpha polypeptide" refers to a fragment of the TCR alpha chain or a homolog thereof that comprises an extracellular domain lacking an antigen-binding domain (i.e. lacking a variable domain), a transmembrane domain, and optionally an intracellular domain, and that maintains at least the ability of the TCR alpha chain to be presented as a TCR complex on the surface of a T cell that expresses it.

[0091] As used herein, "TCR alpha" or "TCR alpha chain" refers to the polypeptide expression product of the TRA gene (corresponding to human gene ID 6955) or a homologue thereof. According to certain embodiments, TCR alpha refers to human TCR alpha. According to certain embodiments, TCR alpha refers to mouse TCR alpha.

[0092] According to certain embodiments, the TCR alpha polypeptide comprises the amino acid sequence of a TCR alpha constant domain or a fragment or homologue thereof, a TCR alpha hinge region, a TCR alpha transmembrane domain, and a TCR alpha intracellular domain.

[0093] According to specific embodiments, the TCR alpha polypeptide is less than 170, less than 160, less than 155, or less than 152 amino acids in length, each possibility representing a separate embodiment of the present invention.

[0094] According to specific embodiments, the TCR alpha polypeptide is at least 120, at least 125, at least 130, at least 135, at least 140, or at least 150 amino acids in length, each possibility representing a separate embodiment of the present invention.

[0095] According to a particular embodiment, the TCR alpha polypeptide is 135-151 amino acids in length.

[0096] According to a particular embodiment, the TCR alpha polypeptide is about 135 amino acids in length.

[0097] According to a particular embodiment, the TCR alpha polypeptide is 140-151 amino acids in length.

[0098] According to a particular embodiment, the TCR alpha polypeptide is about 141 amino acids in length.

[0099] According to a particular embodiment, the TCR alpha polypeptide is about 151 amino acids in length.

[0100] According to certain embodiments, the TCR alpha polypeptide is about 145 amino acids in length.

[0101] According to certain embodiments, the TCR alpha polypeptide is about 150 amino acids in length.

[0102] According to certain embodiments, the TCR alpha constant domain of the TCR alpha polypeptide is at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90 amino acids in length.

[0103] According to certain embodiments, the TCR alpha constant domain of the TCR alpha polypeptide is at least 84 amino acids in length.

[0104] According to certain embodiments, the TCR alpha constant domain of the TCR alpha polypeptide is at least 88 amino acids in length.

[0105] According to a particular embodiment, the TCR alpha constant domain of the TCR alpha polypeptide is 94 amino acids in length.

[0106] According to certain embodiments, the TCR alpha constant domain of the TCR alpha polypeptide is at least the sequence of amino acid 11 of SEQ ID NO:9 to amino acid 94 of SEQ ID NO:9, although homologous sequences and sequence modifications of SEQ ID NO:9 are further contemplated as further described herein above and below.

[0107] According to certain embodiments, the TCR alpha constant domain of the TCR alpha polypeptide is at least the sequence between amino acid 7 of SEQ ID NO:9 and amino acid 94 of SEQ ID NO:9, although homologous sequences and sequence modifications of SEQ ID NO:9 are further contemplated as further described above and below.

[0108] According to certain embodiments, the TCR alpha peptide comprises the entire constant domain of TCR alpha.

[0109] According to certain embodiments, the TCR alpha polypeptide comprises a mouse TCR alpha polypeptide.

[0110] A non-limiting example of a mouse TCR alpha polypeptide that can be used in certain embodiments of the invention is provided in Uniprot ID A0A075B662 (SEQ ID NO:8).

[0111] According to certain embodiments, the TCR alpha polypeptide comprises a human TCR alpha polypeptide.

[0112] A non-limiting example of a human TCR alpha polypeptide lacking a variable domain is provided in Uniprot ID P01848 (SEQ ID NO:9).

[0113] As used herein, "TCR beta polypeptide" refers to a fragment of a TCR beta chain or a homolog thereof that comprises an extracellular domain lacking an antigen-binding domain (i.e. lacking a variable domain), a transmembrane domain, and optionally an intracellular domain, and that maintains at least the ability of the TCR beta chain to be presented as a TCR complex on the surface of a T cell that expresses it.

[0114] As used herein, "TCR beta" or "TCR beta chain" refers to the polypeptide expression product of the TRB gene (corresponding to human gene ID 6957) or homologs thereof, including TRBC1 (corresponding to human gene ID 28639) and TRBC2 (corresponding to human gene ID 28638) or homologs thereof.

[0115] According to a particular embodiment, TCRβ refers to TRBC1.

[0116] According to a particular embodiment, TCRβ refers to TRBC2.

[0117] According to certain embodiments, TCRβ refers to human TCRβ.

[0118] According to a particular embodiment, TCRβ refers to mouse TCRβ.

[0119] According to certain embodiments, the TCR β polypeptide comprises the amino acid sequence of a TCR β constant domain or a fragment or homologue thereof, a TCR β hinge region, a TCR β transmembrane domain, and a TCR β intracellular domain.

[0120] According to specific embodiments, the TCR β polypeptide is less than 250, less than 200, less than 190, or less than 188 amino acids in length, each possibility representing a separate embodiment of the present invention.

[0121] According to specific embodiments, the TCR β polypeptide is at least 140, at least 150, at least 160, at least 170, at least 180, or at least 185 amino acids in length, each possibility representing a separate embodiment of the present invention.

[0122] According to certain embodiments, the TCR β polypeptide is 170-187 amino acids in length.

[0123] According to certain embodiments, the TCR β polypeptide is about 172 amino acids in length.

[0124] According to certain embodiments, the TCR β polypeptide is about 178 amino acids in length.

[0125] According to certain embodiments, the TCR β polypeptide is approximately 183 amino acids in length.

[0126] According to certain embodiments, the TCR β polypeptide is about 187 amino acids in length.

[0127] According to certain embodiments, the TCR β constant domain of the TCR β polypeptide is at least 100, at least 110, at least 120, at least 125 amino acids in length.

[0128] According to a particular embodiment, the TCR β constant domain of the TCR β polypeptide is 130 amino acids in length.

[0129] According to certain embodiments, the TCR β peptide comprises the entire constant domain of TCR β.

[0130] According to certain embodiments, the TCR β polypeptide comprises a mouse TCR β polypeptide.

[0131] Non-limiting examples of mouse TCR β polypeptides that may be used with certain embodiments of the present invention are provided in SEQ ID NOs: 10-11.

[0132] According to certain embodiments, the TCR β polypeptide comprises a human TCR β polypeptide.

[0133] Non-limiting examples of human TCR β polypeptides lacking variable domains are provided in SEQ ID NOs:12-13.

[0134] To allow the presentation of TCRs with human TCR alpha and beta polypeptides lacking binding domains (antigen binding domain, heterologous extracellular binding domain), the inventors envisaged modifications in the sequences of the human TCR alpha and beta polypeptides. Non-limiting examples of such modifications include the insertion of a heterologous dimerization moiety and the insertion of a minimal mouse amino acid sequence.

[0135] Thus, according to certain embodiments, the TCR alpha and / or beta polypeptides include chimeric human and mouse TCR alpha and / or beta polypeptides. Thus, for example, the TCR alpha and / or beta polypeptides may be based on human TCR alpha and / or beta that contain a short (e.g., 3-20) mouse amino acid sequence. Such sequences are known in the art and described, for example, in Sommermeyer D et al., J Immunol. 2010; Bialer G et al., J Immunol. 2010, the contents of which are fully incorporated herein by reference.

[0136] Thus, according to certain embodiments, the TCR alpha polypeptide comprises a human TCR alpha polypeptide comprising the amino acid substitutions (or changes or mutations) P91S, E92D, S93V, and S94P corresponding to the TCR alpha amino acid sequence set forth in SEQ ID NO:9.

[0137] According to certain embodiments, the TCR β polypeptide comprises a human TCR β polypeptide comprising the amino acid substitutions (or changes or mutations) E18K, S22A, F133I, E / V136A, Q139H corresponding to the TCR β amino acid sequence set forth in SEQ ID NO:12.

[0138] As used herein, the phrase "corresponding to SEQ ID NO:9" is intended to include the corresponding amino acid residues for any other TCR alpha amino acid sequence.

[0139] As used herein, the phrase "corresponding to SEQ ID NO: 12" is intended to include the corresponding amino acid residues for any other TCR β amino acid sequence.

[0140] Thus, according to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:14 and the TCR beta polypeptide comprises SEQ ID NO:15 or SEQ ID NO:16.

[0141] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:111 and the TCR beta polypeptide comprises SEQ ID NO:112 or 113.

[0142] According to certain embodiments, the TCR alpha and / or beta polypeptides may be based on human TCR alpha and / or beta including the hinge region of mouse TCR alpha and / or beta, respectively.

[0143] According to certain embodiments, the TCR alpha and / or beta polypeptides may comprise the transmembrane domain and optionally the intracellular domain of human TCR alpha and / or beta, respectively, and the extracellular domain of mouse TCR alpha and / or beta, respectively.

[0144] According to certain embodiments, the TCR comprises a heterodimerization moiety.

[0145] According to certain embodiments, the heterodimerization moiety is located in the extracellular domain of the TCR.

[0146] According to a particular embodiment, the heterodimerization moiety is located in the constant domain of the TCR.

[0147] According to a particular embodiment, the heterodimerization moiety is located in the hinge domain of the TCR.

[0148] As used herein, the term "dimerization moiety" refers to a heterologous amino acid sequence capable of forming a TCRα polypeptide-TCRβ polypeptide heterodimer. Such amino acids include, for example, amino acid sequences that include at least two cysteine ​​residues (one in the TCRα polypeptide and a second cysteine ​​residue in the TCRβ polypeptide), allowing the formation of disulfide bonds between thiol groups. Another example is a dimerization domain or an amino acid sequence that allows the three-dimensional formation of a dimer. Such sequences are known to those skilled in the art. Methods for determining dimerization are known in the art and include, but are not limited to, immunoprecipitation, size exclusion chromatography, fast protein liquid chromatography (FPLC), multi-angle light scattering (SEC-MALS) analysis, SDS-PAGE analysis, nano-DSF, yeast two-hybrid system (e.g., RRS), and flow cytometry.

[0149] Any known dimerization moiety known in the art can be used in certain embodiments of the invention. Non-limiting examples of dimerization moieties that can be used with certain embodiments of the invention include heterologous cysteine ​​residues in each of the TCR alpha and beta polypeptides, the Fc domain of an antibody (not including the antibody antigen-binding domain), mouse amino acid sequences capable of forming heterodimers, such as the short mouse amino acid sequences described herein above, FRB-FKBP, leucine zippers.

[0150] According to certain embodiments, each of the TCR α and β polypeptides comprises at least one heterologous cysteine.

[0151] According to certain embodiments, the heterologous cysteines are located in the extracellular domain of each of the TCR α and β polypeptides.

[0152] According to certain embodiments, the heterologous cysteines are located in the hinge or constant domain of each of the TCR α and β polypeptides.

[0153] According to certain embodiments, the TCR alpha polypeptide comprises a T48C amino acid substitution corresponding to the TCR alpha amino acid sequence set forth in SEQ ID NO: 9, and the TCR beta polypeptide comprises an S57C amino acid substitution corresponding to the TCR beta amino acid sequence set forth in SEQ ID NO: 12.

[0154] Thus, according to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:17 and the TCR beta polypeptide comprises SEQ ID NO:18 or SEQ ID NO:19.

[0155] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:114 and the TCR beta polypeptide comprises SEQ ID NO:115 or 116.

[0156] Because each of the TCR α and β polypeptides contains an endogenous cysteine ​​located in the hinge region of the polypeptide that forms a single disulfide bond between the α and β polypeptides, the addition of at least one heterologous cysteine ​​results in the presence of at least two cysteine ​​residues in each of the TCR α and β polypeptides that allow for the formation of at least two disulfide bonds between the TCR α and TCR β polypeptides.

[0157] Thus, according to certain embodiments, the TCR α and β polypeptides each contain at least two cysteine ​​residues capable of forming at least two disulfide bonds between the α and β polypeptides.

[0158] According to certain embodiments, the at least two cysteine ​​residues are located in the extracellular domain of each of the TCR α and β polypeptides.

[0159] According to certain embodiments, at least one cysteine ​​residue is located in the hinge region of each of the TCR α and β polypeptides, and at least one cysteine ​​residue is located in the hinge or constant domain of each of the TCR α and β polypeptides.

[0160] The terms "TCR alpha polypeptide" and "TCR beta polypeptide" also encompass functional homologs (naturally occurring or synthetically / recombinantly produced) that exhibit the desired activity (i.e., are presented as a TCR complex on the surface of a T cell that expresses it). Such homologues may be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or homologous to the polypeptides set forth in SEQ ID NOs: 8, 9, 14, 17, 111, or 114 for TCR alpha and SEQ ID NOs: 10-13, 15-16, 18-19, 112-113, or 115-116 for TCR beta (as described further below).

[0161] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0162] Homologs can also refer to orthologs, deletions, insertions, or substitution variants including amino acid substitutions, as further described below.

[0163] According to certain embodiments, the TCR alpha and / or beta polypeptides may contain conservative and / or non-conservative amino acid substitutions. Non-limiting examples of such substitutions are known in the art and are described, for example, in Haga-Friedman A et al., J Immunol. 2012;188:5538-46; Froning K et al., Nat Commun [Internet]. Springer US; 2020;11:1-14; Boulter JM et al., Protein Eng. 2003;16:707-11, the contents of which are fully incorporated herein by reference.

[0164] According to certain embodiments, the TCR alpha and / or TCR beta polypeptides do not comprise conservative and / or non-conservative amino acid substitutions at endogenous cysteine ​​residues, for example endogenous cysteine ​​residues located in the hinge regions of each of the TCR alpha and beta.

[0165] The term "conservative substitution" as used herein refers to the replacement of an amino acid present in a natural sequence in a peptide with a natural or non-natural amino acid or a peptidomimetic having similar steric properties. If the side chain of the natural amino acid being substituted is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptidomimetic moiety that is polar or hydrophobic (in addition to having the same steric properties as the side chain of the substituted amino acid).

[0166] Since naturally occurring amino acids are typically classified according to their properties, conservative substitutions with naturally occurring amino acids can be readily determined, taking into account the fact that, according to the present invention, the replacement of a charged amino acid with a sterically similar uncharged amino acid is considered a conservative substitution.

[0167] It is also possible to use amino acid analogs (synthetic amino acids) well known in the art to generate conservative substitutions with non-naturally occurring amino acids. Peptide mimetics of naturally occurring amino acids are well described in the literature known to those skilled in the art.

[0168] When affecting a conservative substitution, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

[0169] The phrase "non-conservative substitution," as used herein, refers to the replacement of an amino acid present in a parent sequence with another natural or unnatural amino acid that has different electrochemical and / or steric properties. Thus, the side chain of the substituted amino acid can be significantly larger (or smaller) than the side chain of the natural amino acid being substituted and / or can have a functional group that has significantly different electronic properties than the amino acid being substituted. Examples of this type of non-conservative substitution include the substitution of phenylalanine or cyclohexylmethylglycine for alanine, isoleucine for glycine, or -NH-CH[(-CH 2 ) 5 These non-conservative substitutions that fall within the scope of the present invention still constitute peptides that have neuroprotective properties.

[0170] Thus, according to certain embodiments, the one or more amino acid substitutions (or changes or mutations) in the TCR alpha are selected from the group consisting of S116L, G119V, and F120L, which correspond to the TCR alpha amino acid sequence set forth in SEQ ID NO:9.

[0171] According to certain embodiments, the TCR alpha polypeptide comprises the amino acid substitutions S116L, G119V, and F120L corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO:9.

[0172] According to certain embodiments, the TCR alpha polypeptide comprises the amino acid substitutions T48C, S116L, G119V, and F120L corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and the TCR beta polypeptide comprises the amino acid substitution S57C corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12.

[0173] According to certain embodiments, the TCR alpha polypeptide and the TCR beta polypeptide comprise chimeric human and mouse TCR alpha polypeptides and chimeric human and mouse TCR beta polypeptides, further comprising the amino acid substitution T48C corresponding to the human TCR alpha polypeptide set forth in SEQ ID NO: 9, and the TCR beta polypeptide comprises the amino acid substitution S57C corresponding to the human TCR beta polypeptide set forth in SEQ ID NO: 12.

[0174] According to certain embodiments, the one or more amino acid changes in the TCR alpha are selected from the group consisting of S21F, T32I, A72T, which correspond to the TCR alpha amino acid sequence set forth in SEQ ID NO:9.

[0175] According to other specific embodiments, the TCR alpha polypeptide does not contain one or more amino acid changes selected from the group consisting of S21F, T32I, A72T corresponding to the TCR alpha amino acid sequence set forth in SEQ ID NO:9.

[0176] According to certain embodiments, the one or more amino acid changes in the TCR β are selected from the group consisting of E18K, H23R, D39P, S54D, which correspond to the TCR β amino acid sequence set forth in SEQ ID NO:12.

[0177] According to other specific embodiments, the TCR β polypeptide does not contain one or more amino acid changes selected from the group consisting of E18K, H23R, D39P, S54D corresponding to the TCR β amino acid sequence set forth in SEQ ID NO:12.

[0178] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 17, 20-23, 47, 111, 114, and 117-120, with each possibility representing a separate embodiment of the present invention.

[0179] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 17, 20-23, 47, 111, 114, and 117-120.

[0180] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 17, and 20-23, with each possibility representing a separate embodiment of the present invention.

[0181] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 14, 17, and 20-23.

[0182] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20-23, 47, 111, 114, and 117-120, with each possibility representing a separate embodiment of the present invention.

[0183] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20-23, 47, 111, 114, and 117-120.

[0184] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, and 20-23, with each possibility representing a separate embodiment of the present invention.

[0185] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, and 20-23.

[0186] According to certain embodiments, the TCR alpha polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 20-23, with each possibility representing a separate embodiment of the present invention.

[0187] According to certain embodiments, the TCR α polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 20-23.

[0188] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-11, 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122, with each possibility representing a separate embodiment of the present invention.

[0189] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-11, 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122.

[0190] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-11, 15-16, 18-19, and 24-25, with each possibility representing a separate embodiment of the present invention.

[0191] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-11, 15-16, 18-19, and 24-25.

[0192] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122, with each possibility representing a separate embodiment of the present invention.

[0193] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122.

[0194] According to certain embodiments, the TCR β polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, and 24-25, with each possibility representing a separate embodiment of the present invention.

[0195] According to a particular embodiment, the TCR β polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19 and 24-25.

[0196] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:8 and the TCR beta polypeptide comprises SEQ ID NO:10 or SEQ ID NO:11.

[0197] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:14 and the TCR beta polypeptide comprises SEQ ID NO:15 or SEQ ID NO:16.

[0198] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:111 and the TCR beta polypeptide comprises SEQ ID NO:112 or 113.

[0199] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO: 17 or 20, and the TCR beta polypeptide comprises SEQ ID NO: 18 or 19.

[0200] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO: 114 or 117, and the TCR beta polypeptide comprises SEQ ID NO: 115 or 116.

[0201] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO: 21 or 23, and the TCR beta polypeptide comprises SEQ ID NO: 24 or 25.

[0202] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO: 118 or 120, and the TCR beta polypeptide comprises SEQ ID NO: 121 or 122.

[0203] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:22 and the TCR beta polypeptide comprises SEQ ID NO:15 or SEQ ID NO:16.

[0204] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:119 and the TCR beta polypeptide comprises SEQ ID NO:112 or 113.

[0205] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:47 and the TCR beta polypeptide comprises SEQ ID NO:18 or SEQ ID NO:19.

[0206] According to certain embodiments, the TCR alpha polypeptide comprises SEQ ID NO:47 and the TCR beta polypeptide comprises SEQ ID NO:115 or 116.

[0207] According to certain embodiments, the TCR lacks an extracellular domain heterologous to the TCR alpha and beta polypeptides defined herein.

[0208] However, according to certain embodiments, the TCR may include a heterologous amino acid sequence translationally fused to the TCR alpha and / or beta polypeptide, so long as it is not an extracellular domain (e.g., an extracellular binding domain). Non-limiting examples of such heterologous sequences may include amino acid sequences such as trafficking sequences (e.g., CD8 membrane transport peptide as provided in SEQ ID NO: 42), intracellular domains such as tags, signaling domains, and the like.

[0209] According to certain embodiments, the TCR may include a heterologous amino acid sequence translationally fused to the TCR alpha and / or beta polypeptide, so long as it is not an antigen binding domain. Non-limiting examples of such heterologous sequences may include amino acid sequences of trafficking sequences (e.g., CD8 membrane transport peptide as provided in SEQ ID NO: 42), receptors, ligands or hormones, signaling domains.

[0210] In certain embodiments, the TCR comprises a heterologous domain of a receptor or ligand. Non-limiting examples of such receptors include EGFR, HER2, VEGFR1, VEGFR2, EpoR, FGFR1, FGFR2, FGFR3, FGFR4, MPL, CSF3R, CSF2RA, FLT3, CD117, PD1, CTLA4, CD28, designed ankyrin repeat proteins (DARPins). Non-limiting examples of such ligands include TPO, IL2, IL15, IL18, SCF.

[0211] According to certain embodiments, the TCR lacks a heterologous domain of the receptor or ligand.

[0212] According to certain embodiments, the TCR comprises a heterologous signaling domain, which may be an activating or inhibitory signaling domain.

[0213] As used herein, the phrase "activation signal" refers to an amino acid sequence capable of transmitting a primary or costimulatory signal that results in T cell proliferation, maturation, cytokine production, and / or induction of regulatory or effector functions. Typically, an activation primary stimulatory signal domain contains an ITAM domain, and a costimulatory signal domain does not contain an ITAM domain.

[0214] Any known activation signal domain can be used in certain embodiments of the present invention. Non-limiting examples of activation signaling domains include the intracellular signaling domains of proteins CD3 zeta, FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, CD28, OX40, ICOS, CD27, ICOS, GITR, HVEM, TIM1, LFA1 (CD11a), CD2, CD40L, LIGHT, CD30, Fc receptor, DAP10, and DAP12.

[0215] As used herein, the phrase "inhibitory signal" refers to an amino acid sequence capable of transmitting a primary or secondary inhibitory signal that results in the suppression of T cell proliferation, maturation, cytokine production and / or induction of regulatory or effector functions. According to certain embodiments, the inhibitory signal domain is an intracellular domain comprising an ITIM domain.

[0216] Any known inhibitory signaling domain may be used in certain embodiments of the invention. Non-limiting examples of inhibitory signaling domains include the intracellular signaling domains of the proteins PD1, CTLA4, LAG3, TIM3, BTLA, TIGIT, 2B4, CD300LF, PECAM, LY9, SIRPA, and CD244.

[0217] Methods for determining the signal transduction of activation or inhibitory signals in T cells are well known in the art and include, but are not limited to, binding assays using, for example, BiaCore, HPLC, or flow cytometry, enzyme activity assays such as kinase activity assays, and expression of molecules involved in signal transduction cascades using, for example, PCR, Western blot, immunoprecipitation, and immunohistochemistry. Additionally or alternatively, determining the transmission of signals can be achieved by assessing T cell activation or function. Methods for assessing T cell activation or function are well known in the art and include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, expression of activation markers such as CD25, CD69, and CD69 using flow cytometry.

[0218] According to other particular embodiments, the TCR lacks a heterologous signaling domain.

[0219] According to a particular embodiment, the TCR comprises or is bound to a heterologous tag.

[0220] According to certain embodiments, the tag is a detectable moiety.

[0221] Examples of detectable moieties that can be used in the present invention include radioisotopes (e.g.,

[0125] iodine), phosphorescent chemiluminescent or fluorescent chemicals and enzymes (e.g., horseradish peroxidase (HPR), β-galactosidase, and alkaline phosphatase (AP)). Further examples of detectable moieties include those detectable by positron emission tomography (PET) and magnetic resonance imaging (MRI), all of which are well known to those of skill in the art.

[0222] Non-limiting examples of tags that can be used in certain embodiments include chitin-binding protein (CBP) tags, maltose-binding protein (MBP) tags, glutathione-S-transferase (GST) tags, poly(His) tags, FLAG tags, biotin, histidine, epitope tags, such as V5 tags, c-myc tags, and HA tags, as well as fluorescent tags, such as green fluorescent protein (GFP or EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and cyan fluorescent protein (CFP), as well as derivatives of these tags, or any tag known in the art.

[0223] Non-limiting schematics and sequences of TCRs of some embodiments of the present invention are provided in Figures 2A-B and SEQ ID NOs: 2, 26-32, 46, 49, 63-68, and 125.

[0224] Typically, the TCRs disclosed herein are produced by recombinant DNA techniques.

[0225] Thus, according to one aspect of the present invention there is provided at least one polynucleotide encoding a TCR or a TCR complex.

[0226] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence, and / or a composite polynucleotide sequence (e.g., a combination of the above).

[0227] According to certain embodiments, the at least one polynucleotide comprises a nucleic acid sequence encoding a TCR alpha polypeptide and a nucleic acid sequence encoding a TCR beta polypeptide.

[0228] According to certain embodiments, the TCR alpha and beta polypeptides are encoded by a single polynucleotide. Further description of the expression of multiple polypeptides from a single polynucleotide is provided below.

[0229] Thus, according to a particular embodiment, the at least one polynucleotide is one polynucleotide.

[0230] According to other specific embodiments, separate polynucleotides are used to encode the TCR α and β polypeptides.

[0231] Thus, according to a particular embodiment, the at least one polynucleotide is at least two polynucleotides.

[0232] According to a particular embodiment, the at least one polynucleotide is two polynucleotides.

[0233] According to a particular embodiment, the at least one polynucleotide further comprises a nucleic acid sequence encoding a CD3 polypeptide.

[0234] In order to express any of the disclosed polypeptides in a cell, the polynucleotide sequence encoding the polypeptide is preferably linked to a nucleic acid construct suitable for cell expression. Such a nucleic acid construct comprises at least one cis-acting regulatory element for directing the expression of the nucleic acid sequence. Cis-acting regulatory sequences include those that direct the constitutive expression of a nucleotide sequence, as well as those that direct the inducible expression of a nucleotide sequence only under certain conditions. Thus, for example, a promoter sequence is included in the nucleic acid construct for directing the transcription of the polynucleotide sequence in a cell in a constitutive or inducible manner.

[0235] The nucleic acid constructs (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences (e.g., shuttle vectors) that render the vector suitable for replication and integration. In addition, a typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and a polyadenylation signal. By way of example, such constructs typically contain a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof.

[0236] The nucleic acid constructs of some embodiments of the invention typically comprise or encode a signal sequence for targeting the polypeptide to the cell surface. According to certain embodiments, the signal sequence for this purpose is a mammalian signal sequence or a signal sequence of the polypeptide variant of some embodiments of the invention.

[0237] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription start site, is thought to be involved in directing RNA polymerase to initiate RNA synthesis. Other upstream promoter elements determine the rate at which transcription is initiated.

[0238] Preferably, the promoters utilized by the nucleic acid constructs of some embodiments of the present invention are active in a particular transformed cell population, namely T cells. Examples of T cell specific promoters include lymphoid specific promoters [Calame et al. (1988) Adv. Immunol. 43:235-275]; in particular, the promoter of the T cell receptor [Winoto et al. (1989) EMBO J. 8:729-733].

[0239] Enhancer elements can stimulate transcription up to 1,000-fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the invention include those derived from polyoma virus, human or mouse cytomegalovirus (CMV), long terminal repeat sequences from various retroviruses (e.g., murine leukemia virus, murine or Rous sarcoma virus, and HIV). See Enhancer and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1983, incorporated herein by reference.

[0240] In constructing an expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting, although, as is known in the art, some variation in this distance can be accommodated without loss of promoter function.

[0241] Polyadenylation sequences may also be added to expression vectors to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: a GU- or U-rich sequence located downstream from the polyadenylation site, and a highly conserved sequence of 6 nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals suitable for some embodiments of the invention include those derived from SV40.

[0242] In addition to the elements already described, the expression vectors of some embodiments of the present invention may typically contain other specialized elements intended to increase the level of expression of the cloned nucleic acid or to facilitate the identification of cells carrying the recombinant DNA. For example, many animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate episomally as long as the appropriate factors are provided by genes carried on the plasmid or with the genome of the host cell.

[0243] Vector may or may not contain eukaryotic replicon.If eukaryotic replicon exists, vector can be amplified in eukaryotic cell by using suitable selection marker.If vector does not contain eukaryotic replicon, episomal amplification is not possible.Instead, recombinant DNA is integrated into the genome of engineered cell, where promoter directs the expression of desired nucleic acid.

[0244] Expression vectors of some embodiments of the invention may further comprise additional polynucleotide sequences, e.g., allowing translation of several proteins from a single mRNA, such as an internal ribosome entry site (IRES) or a self-cleavable peptide, e.g., a spacer and / or a protease, e.g., a 2A peptide (e.g., P2A, T2A, E2A) that may be accompanied by a furin cleavage site (see, e.g., Yang et al., Gene Ther. 2008;15(21):1411-1423); and sequences for genomic integration.

[0245] It will be understood that the individual elements contained within an expression vector may be arranged in a variety of configurations. For example, enhancer elements, promoters, etc., and even the polynucleotide sequence(s) encoding a polypeptide may be arranged in a "head-to-tail" configuration, may exist in a complementary configuration as reverse complements, or as antiparallel strands. Although such various configurations are more likely to occur with the non-coding elements of an expression vector, alternative configurations of coding sequences within an expression vector are also envisioned.

[0246] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0247] Expression vectors containing regulatory elements derived from eukaryotic viruses (e.g., retroviruses) can also be used. SV40 vectors include pSVT7 and pMT2. Bovine papilloma virus-derived vectors include pBV-1MTHA, and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A, pAV009 / B, pAV009 / C, pAV009 / D, pAV009 / E, pAV009 / F, pAV009 / G, pAV009 / H, pAV009 / I, pAV009 / L, pAV009 / N ... + , pMTO10 / A + , pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0248] As mentioned above, viruses are often highly specialized infectious agents that have evolved to evade host defense mechanisms. Typically, viruses infect and grow in specific cell types. The target specificity of viral vectors utilizes their natural specificity to specifically target predetermined cell types, thereby introducing recombinant genes into infected cells. The ability to select suitable vectors for transforming T cells is well within the capabilities of those skilled in the art, and therefore a general description of the selection considerations is not provided herein.

[0249] Recombinant viral vectors are useful for in vivo expression of polypeptides because they offer advantages such as lateral infection and target specificity. Lateral infection, for example, is inherent in the life cycle of retroviruses, and is the process by which a single infected cell produces many progeny virions that bud and infect neighboring cells. This results in a large area being rapidly infected, most of which were not initially infected by the original viral particle. This is in contrast to vertical infection, where the infectious agent spreads only through daughter progeny. Viral vectors that cannot spread laterally can also be made. This feature can be useful when the desired objective is to introduce a specific gene into only a localized number of target cells.

[0250] A variety of methods can be used to introduce the expression vectors of some embodiments of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. See also US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0251] Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiencies can be obtained due to the infectivity of the virus.

[0252] Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs (e.g., adenovirus, lentivirus, herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems). Useful lipids for lipid-mediated gene transfer are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1):54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenovirus, AAV, lentivirus, or retrovirus. Viral constructs, such as retroviral constructs, contain at least one transcription promoter / enhancer or locus-defining element, or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA transport, or post-translational modification of messengers. Such vector constructs also include a packaging signal, a long terminal repeat (LTR) or a portion thereof, and a plus strand primer binding site and a minus strand primer binding site appropriate for the virus used, unless already present in the viral construct. Furthermore, such constructs typically include a signal sequence for targeting the polypeptide to a desired site in the cell. According to certain embodiments, the signal sequence includes a membrane transport sequence. Such sequences are known in the art. A non-limiting example of such a sequence is the CD8 signal peptide as provided in SEQ ID NO: 43. Optionally, the construct may also include a signal directing polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof. Other vectors that are non-viral (e.g., cationic lipids, polylysine, and dendrimers) can be used.

[0253] According to certain embodiments, the polynucleotide is expressed in a cell, for example while knocking out the endogenous TCR (i.e., knock-in / knock-out). Such methods are known in the art [e.g., Menke D. Genesis (2013) 51:-618; Capecchi, Science (1989) 244:1288-1292; Santiago et al., Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO2014085593, WO2009071334, and WO2011146121; U.S. Patent Nos. 8,771,945, 8,586,526, 6,774,279, and U.S. Patent Application Publication Nos. 20030232410, 20050026157, and 20060014264; the contents of which are incorporated by reference in their entireties], targeted homologous recombination, site-specific recombinases, PB transposases, and genome editing with engineered nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas systems).

[0254] Non-limiting schematic diagrams and nucleic acid constructs encoding TCRs of some embodiments of the invention are provided in Figures 2A-B and SEQ ID NOs: 33-40, 53, 73-79, and 126.

[0255] Certain embodiments of the invention also contemplate cells, e.g., T cells, that comprise a TCR or TCR complex as described herein, and methods of producing the same.

[0256] Thus, according to one aspect of the invention there is provided a transduced cell expressing a TCR complex or a TCR or at least one polynucleotide encoding same.

[0257] According to one aspect of the invention there is provided a transduced T cell expressing a TCR complex or a TCR or at least one polynucleotide encoding same.

[0258] According to further or alternative aspects of the invention there is provided a method of producing a TCR or TCR complex expressing cell, the method comprising introducing into a cell at least one polynucleotide encoding a TCR as disclosed herein under conditions allowing expression of the TCR or TCR complex.

[0259] According to further or alternative aspects of the invention there is provided a method of producing a TCR or TCR complex-expressing T cell, the method comprising introducing into a T cell at least one polynucleotide encoding a TCR as disclosed herein under conditions allowing expression of the TCR or TCR complex.

[0260] According to further or alternative aspects of the invention there is provided a method of expressing a TCR or TCR complex in a T cell, the method comprising introducing into a T cell at least one polynucleotide encoding a TCR as disclosed herein under conditions allowing expression of the TCR or TCR complex.

[0261] Such conditions include, for example, a suitable temperature (e.g., 37° C.), atmosphere (e.g., air + 5% CO 2 ), pH, light, medium, supplements, etc.

[0262] According to other particular embodiments, the introducing is performed in vivo.

[0263] According to certain embodiments, the introduction is performed in vitro or ex vivo.

[0264] Non-limiting examples of cells into which a polynucleotide can be introduced include immune cells, such as T cells, pluripotent stem cells, hematopoietic stem cells, and progenitor cells.

[0265] Non-limiting examples of stem cells that can be used in certain embodiments of the present invention include embryonic stem (ES) cells, germline stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, umbilical cord blood-derived pluripotent stem cells, bone marrow-derived stem cells, and the like.

[0266] According to certain embodiments, the cell into which the polynucleotide is introduced is an iPS cell.

[0267] According to certain embodiments, the stem or progenitor cells can be further differentiated after introduction, for example into T cells, thereby obtaining T cells expressing the TCR disclosed herein. Methods for differentiating stem or progenitor cells into T cells are known in the art and are disclosed, for example, in Blood, 105(4):1431-1439, 2005; and International Patent Publication Nos. WO2016 / 076415 and WO2017 / 221975.

[0268] According to a particular embodiment, the cell into which the polynucleotide is introduced is a T cell.

[0269] As used herein, the term "T cells" refers to differentiated lymphocytes that express CD3, and includes CD4+ cells, CD8+ cells, and NKT cells.

[0270] According to certain embodiments, the T cells are effector cells.

[0271] As used herein, the term "effector T cell" refers to a T cell that activates or directs other immune cells, e.g., by producing cytokines, or has cytotoxic activity, e.g., CD4+, Th1 / Th2, CD8+ cytotoxic T lymphocytes.

[0272] According to certain embodiments, the T cells are regulatory cells.

[0273] As used herein, the term "regulatory T cells" or "Treg" refers to T cells that negatively regulate the activation of effector T cells as well as other T cells, including cells of the innate immune system. Treg cells are characterized by a sustained suppression of effector T cell responses. According to certain embodiments, Treg are CD4+CD25+Foxp3+ T cells.

[0274] According to a particular embodiment, the T cells are CD4+ T cells.

[0275] According to other specific embodiments, the T cells are CD8+ T cells.

[0276] According to certain embodiments, the T cells are naive T cells.

[0277] According to certain embodiments, the T cells are memory T cells. Non-limiting examples of memory T cells include effector memory CD4+ T cells with a CD3+ / CD4+ / CD45RA- / CCR7- phenotype, central memory CD4+ T cells with a CD3+ / CD4+ / CD45RA- / CCR7+ phenotype, effector memory CD8+ T cells with a CD3+ / CD8+CD45RA- / CCR7- phenotype, and central memory CD8+ T cells with a CD3+ / CD8+CD45RA- / CCR7+ phenotype.

[0278] According to a particular embodiment, the T cells are NKT cells.

[0279] As used herein, the term "NKT cells" refers to specialized T cells that express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8- cells.

[0280] According to other specific embodiments, the T cells are not NKT cells.

[0281] Methods for obtaining T cells are well known in the art. Thus, for example, PBMCs can be isolated by drawing whole blood from a subject, collecting in a container containing an anticoagulant (e.g., heparin or citrate); and apheresis. According to other specific embodiments, T cells are obtained from tissues that contain cells associated with the pathology. Methods for obtaining tissue samples from a subject are well known in the art, and include, for example, biopsy, surgery or autopsy, and preparing a single cell suspension thereof. Then, according to certain embodiments, T cells are enriched or purified from peripheral blood or the single cell suspension. There are several methods and reagents known to those skilled in the art for purifying T cells, such as leukapheresis, sedimentation, density gradient centrifugation (e.g., Ficoll), centrifugal elutriation, fractionation, e.g., chemical lysis of red blood cells (e.g., by ACK), selection of T cells using cell surface markers (e.g., using a FACS sorter or magnetic cell separation techniques, such as those commercially available from, e.g., Invitrogen, Stemcell Technologies, Cellpro, Advanced Magnetics, or Miltenyi Biotec), and depletion of other cell types by methods such as eradication (e.g., killing) with specific antibodies, or by affinity-based purification based on negative selection (e.g., using magnetic cell separation techniques, FACS sorters, and / or capture ELISA labels). Such methods are described, for example, in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, volumes 1-4 (ed. D. N. Weir) and FLOW CYTOMETRY AND CELL SORTING (ed. A. Radbruch, Springer Verlag, 2000).

[0282] According to certain embodiments, the cell, e.g., a T cell, is a mammalian cell.

[0283] According to certain embodiments, the cells, such as T cells, are human cells.

[0284] According to certain embodiments, the cells, e.g., T cells, are from a healthy subject.

[0285] According to certain embodiments, the cells, e.g., T cells, are from a subject suffering from a pathology (e.g., cancer).

[0286] According to a specific embodiment, the cell, eg, a T cell, expresses endogenous CD3.

[0287] According to certain embodiments, the cells, eg, T cells, express exogenous CD3.

[0288] Cells, e.g., T cells, in some embodiments of the invention, are modified (or engineered or transduced) to upregulate or downregulate expression of a gene of interest (e.g., endogenous TCR, PD1, TGFBR1, B2M, CTLA4).

[0289] According to specific embodiments, the cell, e.g., a T cell, does not express an endogenous TCR.

[0290] Thus, according to certain embodiments, the method further comprises downregulating expression of an endogenous TCR, which may be performed before or after introducing at least one polynucleotide encoding a TCR as disclosed herein into the cell.

[0291] According to specific embodiments, the method further comprises downregulating expression of an endogenous TCR prior to introducing into the cell, e.g., T cell, at least one polynucleotide encoding a TCR disclosed herein.

[0292] Methods for downregulating the expression of a gene of interest, such as an endogenous TCR, are well known in the art (see, for example, International Publication No. 2019222275, International Publication No. 2015143224, US Patent No. 20190388472, the contents of which are incorporated by reference in their entirety), including targeted homologous recombination, site-specific recombinase, PB transposase and engineered nuclease-based genome editing (zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR / Cas system, and constant shRNA). Agents for introducing nucleic acid modifications into TCR can be designed from publicly available sources or commercially available from Transposagen, Addgene, and Sangamo Biosciences. Non-limiting examples of methods for downregulating endogenous TCR are described in detail in the Examples section below.

[0293] Cells, e.g., T cells, in some embodiments of the present invention, are modified (or engineered or transduced) to express a chimeric receptor, e.g., a chimeric antigen receptor (CAR).

[0294] As used herein, the phrases "transduced with a CAR" or "engineered to express a CAR" refer to the cloning of a nucleic acid sequence encoding a chimeric antigen receptor (CAR), where the CAR comprises an antigen recognition portion of an antibody and a T cell activation portion. A chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide that contains the antigen binding domain of an antibody (e.g., a single chain variable fragment (scFv)) linked to a T cell signaling domain or a T cell activation domain. Methods of transducing with CARs are known in the art and are disclosed, for example, in Davila et al., Oncoimmunology. 2012 Dec. 1;1(9):1577-1583; Wang and Riviere Cancer Gene Ther. 2015 Mar. 22(2):85-94; Maus et al., Blood. 2014 Apr. 24;123(17):2625-35; Porter DL The New England journal of medicine. 2011, 365(8):725-733; Jackson HJ, Nat Rev Clin Oncol. 2016;13(6):370-383; and Globerson-Levin et al., Mol Ther. 2014;22(5):1029-1038. According to certain embodiments, the antigen recognition moiety is specific for the diseased cell.

[0295] According to other specific embodiments, the cells are not transduced (i.e., do not express) the CAR.

[0296] According to specific embodiments, cells, e.g., T cells, can be freshly isolated and stored, e.g., cryopreserved (i.e., frozen), e.g., at liquid nitrogen temperatures at any stage for extended periods (e.g., months, years) for future use, and cell lineages.

[0297] Methods of cryopreservation are generally known to those of skill in the art and are disclosed, for example, in International Patent Application Publication Nos. WO2007054160 and WO2001039594 and U.S. Patent Application Publication No. US20120149108.

[0298] According to certain embodiments, the cells, e.g., T cells, may be stored in a cell bank or a repository or storage facility.

[0299] As a result, the present teachings further suggest, without limitation, the use of the cells, such as T cells and methods disclosed herein, as a source for adoptive cell therapy.

[0300] Thus, according to one aspect of the invention, the cells disclosed herein, such as T cells, are for use in adoptive cell therapy.

[0301] Cells, e.g., T cells, used according to certain embodiments of the invention may be autologous or non-autologous. They may be syngeneic or non-syngeneic to the subject: allogeneic or xenogeneic. Each possibility represents a separate embodiment of the invention.

[0302] According to certain embodiments, the cells are autologous to the subject.

[0303] According to certain embodiments, the cells are non-autologous to the subject.

[0304] According to certain embodiments, the cells are allogeneic to the subject.

[0305] Because the T cells of some embodiments do not express an endogenous TCR and express a TCR or TCR complex that lacks a binding domain (antigen binding domain, xenogeneic extracellular binding domain), the T cells of some embodiments do not stimulate graft-versus-host disease in non-autologous subjects.

[0306] According to specific embodiments, the T cells described herein are cultured, expanded and / or activated ex vivo prior to administration to a subject.

[0307] Methods for culturing, expanding, and activating T cells are well known to those of skill in the art. For example, T cells of some embodiments can be expanded ex vivo in the presence of anti-CD3 antibodies, anti-CD28 antibodies, anti-CD3, and anti-CD28 coated beads (such as CD3CD28 MACSi Beads available from Miltenyi Biotec) in the presence or absence of IL-2.

[0308] Since the TCRs or TCR complexes disclosed herein lack a binding domain (antigen binding domain, heterologous extracellular binding domain), the T cells of some embodiments of the invention can be administered to a subject in combination with a therapeutic composition intended to bind to a target cell (e.g., a pathological cell) on the one hand and to bind to the TCR complex on the other hand, thereby activating the T cell against the target cell.

[0309] Thus, according to one aspect of the present invention there is provided a method of treating a disease associated with a pathological cell in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell as disclosed herein and a therapeutic composition capable of binding to the pathological cell and the TCR complex, thereby treating the disease in the subject.

[0310] According to further or alternative aspects of the invention, there is provided a T cell as disclosed herein and a therapeutic composition capable of binding to a pathological cell and a TCR complex for use in treating a disease associated with a pathological cell in a subject in need thereof.

[0311] As used herein, the term "subject" includes mammals, preferably humans of any age and any sex. According to certain embodiments, the term "subject" refers to a subject suffering from a pathology (disease, disorder, or medical condition). According to certain embodiments, the term encompasses individuals at risk of developing a pathology.

[0312] As used herein, the term "treat" refers to curing, reversing, attenuating, mitigating, minimizing, suppressing, or halting the deleterious effects of a disease or disorder (e.g., cancer). Those skilled in the art will appreciate that a variety of methodologies and assays can be used to assess the development of pathology, as well as a variety of methodologies and assays can be used to assess the reduction, remission, or regression of pathology (e.g., malignant tumors), as discussed below.

[0313] As used herein, the term "prevent" refers to keeping a disease, disorder, or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0314] As used herein, the phrase "disease associated with pathological cells" means that pathological cells drive the onset and / or progression of the disease.

[0315] According to certain embodiments, the disease may benefit from modulating immune cells.

[0316] As used herein, the phrase "diseases that can benefit from modulating immune cells" refers to diseases in which the activity of a subject's immune response may be sufficient to at least ameliorate symptoms of the disease or delay the onset of symptoms, but in which for some reason the activity of the subject's immune response in doing so is less than optimal.

[0317] According to certain embodiments, the disease may benefit from activating immune cells.

[0318] Non-limiting examples of diseases that can benefit from activating immune cells include hyperproliferative diseases, diseases associated with immunosuppression, immunosuppression caused by medications (e.g., mTOR inhibitors, calcineurin inhibitors, steroids), and infectious diseases.

[0319] According to certain embodiments, the disease comprises an infectious disease.

[0320] As used herein, the term "infectious disease" or "infectious disease" refers to a disease induced by a pathogen, such as a viral pathogen, a bacterial pathogen, for example, an intracellular mycobacterial pathogen (e.g., Mycobacterium tuberculosis), an intracellular bacterial pathogen (e.g., Listeria monocytogenes), or an intracellular protozoan pathogen (e.g., Leishmania and Trypanosoma).

[0321] Specific types of viral pathogens that cause infectious diseases include, but are not limited to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, and filoviruses.

[0322] Specific examples of viral infections that may be treated according to certain embodiments of the present invention include, but are not limited to, human immunodeficiency virus (HIV)-induced acquired immune deficiency syndrome (AIDS), influenza, rhinovirus infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A, B, or C virus infection, measles, papilloma virus infection / warts, cytomegalovirus (CMV) infection, herpes simplex virus infection, yellow fever, Ebola virus infection, rabies virus infection, and the like.

[0323] According to certain embodiments, the disease comprises a hyperproliferative disease.

[0324] According to certain embodiments, the hyperproliferative disease comprises sclerosis, fibrosis, idiopathic pulmonary fibrosis, psoriasis, systemic sclerosis / scleroderma, primary biliary cholangitis, primary sclerosing cholangitis, hepatic fibrosis, prevention of radiation-induced pulmonary fibrosis, myelofibrosis, or retroperitoneal fibrosis.

[0325] According to other particular embodiments, the hyperproliferative disease comprises cancer.

[0326] Thus, according to certain embodiments, the pathological cell is a cancerous cell.

[0327] The cancer that may be treated by some embodiments of the present invention may be any solid or non-solid tumor, cancer metastasis, and / or pre-cancer.

[0328] According to certain embodiments, the cancer is a malignant cancer.

[0329] Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and lymphoma. More specific examples of such cancer include tumors of the gastrointestinal tract (colon cancer, rectal cancer, colorectal cancer, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6); colorectal cancer, hereditary nonpolyposis type 7, small and / or large bowel cancer, esophageal cancer, thickening with esophageal cancer, gastric cancer, pancreatic cancer, pancreatic endocrine tumor), endometrial cancer, dermatofibrosarcoma protuberans, gallbladder cancer, biliary tract tumor, prostate cancer, prostate adenocarcinoma, kidney cancer (e.g., Wilms' tumor, type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cell tumor, immature teratoma of the ovary, uterine cancer, epithelial ovarian cancer, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian cancer, serous ovarian cancer, ovarian spinal tumor, cervical cancer, small cell and non-small cell lung cancer, nasopharyngeal carcinoma, breast cancer (e.g., ductal carcinoma, invasive ductal carcinoma, sporadic, breast cancer, breast cancer susceptibility, type 4 breast cancer, type 1 breast cancer, type 3 breast cancer;breast-ovarian cancer), squamous cell carcinoma (e.g., head and neck cancer), neurogenic tumors, astrocytoma, ganglioneuroblastoma, neuroblastoma, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, B cell, Burkitt's, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), glioma, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancies (tumors), various other cancers (e.g., bronchogenic large cell, ductal, Ehrlich ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinous cell, transitional cell, undifferentiated, differentiation, carcinosarcoma, choriocarcinoma, cystadenocarcinoma), ependymoblastoma, epithelioma, erythroleukemia (e.g., Friend's disease, lymphoblastic), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., pleomorphic, astrocytoma), glioma, hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), adrenal tumor, insulinoma, pancreatic islet tumor, keratoma, epithelioid leiomyoma, leiomyosarcoma, leukemia (e.g., acute lymphocytic, acute lymphoblastic, precursor B cell acute lymphocytic, T cell acute lymphoblastic, acute megakaryoblastic, monocytic myeloid, acute myeloid, acute myeloid, eosinophilic acute myeloid, B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic, friend, granulocytic or myeloid, hairy cell, lymphocytic, megakaryoblastic, monocyte, monocyte-macrophage, myeloblastic, myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic, subacute, T cell, lymphoid neoplasms, predisposition to myeloid malignancies, acute non-lymphocytic leukemia), lymphosarcoma, melanoma, breast cancer, mast cell tumor, medulloblastoma, mesothelioma, metastatic tumor, monocytic tumor, multiple myeloma, spinal cord dysplastic syndromes, myeloma, nephroblastoma, neural tissue glial tumors, neural tissue neuronal tumors, schwannoma, neuroblastoma, oligodendroma, osteochondroma, intraosseous myeloma, osteosarcoma (e.g. Ewing), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g. Ewing, histiocytic, Jensen, osteogenic, reticular cell), neurofibroma, subcutaneous tumor, teratocarcinoma (e.g. pluripotent), teratomas, testicular tumors, thymoma and trichomeoblastoma, gastric carcinoma, fibrosarcoma, anaplastic astrocytoma;These include, but are not limited to, multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, Lynch carcinoma family syndrome II, male germ cell tumors, mast cell leukemia, medullary thyroid, multiple meningiomas, endocrine tumor myxosarcoma, paraganglioma, familial achromophilic, pilomatrixoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial rhabdoid tumor, soft tissue sarcoma, and Turcot's syndrome with glioblastoma;

[0330] According to certain embodiments, the cancer is a pre-malignant cancer.

[0331] Precancers are well characterized and known in the art (see, e.g., Berman JJ. and Henson DE, 2003, Classifying the pre-cancers: a metadata approach. BMC Med Inform Decis Mak. 3:8). Examples of precancers include, but are not limited to, acquired minor precancers, acquired major lesions with nuclear atypia, precursor lesions occurring with genetic hyperplastic syndromes that progress to cancer, and acquired diffuse hyperplasia and diffuse metaplasia. Non-limiting examples of minor precancers include HGSIL (high-grade squamous intraepithelial lesions of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, aberrant crypts (of the colon), and PIN (prostatic intraepithelial neoplasia).

[0332] Non-limiting examples of acquired large lesions with nuclear atypia include tubular adenoma, AILD (angioimmunoblastic lymphadenopathy with dysproteinemia), atypical meningioma, gastric polyp, large plaque pseudopsoriasis, myelodysplasia, papillary transitional cell carcinoma, refractory anemia with hyperblastic properties, and Schneider's papilloma. Non-limiting examples of precursor lesions occurring with inherited hyperplastic syndromes that progress to cancer include atypical lentigo syndrome, C-cell adenomatosis, and MEA. Non-limiting examples of acquired diffuse hyperplasia and diffuse metaplasia include Paget's disease of bone and ulcerative colitis.

[0333] According to certain embodiments, the cancer is selected from the group consisting of lymphoma, leukemia, glioblastoma, colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, and skin cancer.

[0334] According to certain embodiments, the disease may benefit from inhibiting immune cells.

[0335] In certain embodiments, the disease is an autoimmune disease, including, but not limited to, cardiovascular, rheumatic, glandular, gastrointestinal, skin, liver, neurological, muscular, renal, reproductive, connective tissue, and systemic diseases.

[0336] Examples of autoimmune cardiovascular diseases include atherosclerosis (Matsuura E et al., Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O, Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A et al., Lupus. 1998;7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki disease (Praprotnik S. et al., Wien Klin Wochenschr. 2000 Aug 25;112(15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb. Hemost.2000;26(2):157), necrotising small angiitis, microscopic polyangiitis, Churg-Strauss syndrome, autoimmune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne(Paris).2000 May;151(3):178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis). 1999;14(4):171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. June 17, 1999;83(12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. April-June 1999;14(2):114;Semple JW. et al., Blood. 1996 May 15;87(10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 January;28(3-4):285; Sallah S. et al., Ann Hematol. 1997 March;74(3):139), cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 October 15;98(8):1709), and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11(1):9).

[0337] Examples of autoimmune rheumatic diseases include, but are not limited to, rheumatoid arthritis (Krenn V. et al., Histol Histopathol, July 2000; 15(3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A. January 18, 1994; 91(2):437), and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3(3):189).

[0338] Examples of autoimmune glandular diseases include pancreatic disease, type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune antisperm infertility, autoimmune prostatitis, and autoimmune polyglandular syndrome type I. Autoimmune diseases include autoimmune diseases, type 1 diabetes (Castano L. and Eisenbarth GS.Ann.Rev.Immunol.8:647;Zimmet P.Diabetes Res Clin Pract October 1996;34 Suppl:S125), autoimmune thyroid disease, Graves' disease (Orgiazzi J.Endocrinol Metab Clin North Am 2000 June;29(2):339;Sakata S. et al. Mol Cell Endocrinol 1993 March;92(1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 15 December 2000;165(12):7262), Hashimoto's thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57(8):1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57(8):1759), ovarian autoimmunity (Garza KM. et al. J Reprod Immunol 1998 Feb;37(2):87), autoimmune antizoospermia (Diekman AB. et al. Am J Reprod Immunol. 2000 Mar;43(3):134), autoimmune prostatitis (Alexander RB. et al. Urology 1997 Dec;50(6):893), and autoimmune polyglandular syndrome type I (Hara T. et al. Blood. 1991 Mar 1;77(5):1127).

[0339] Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 January;23(1):16), celiac disease (Landau YE. and Shoenfeld Y, 2000 January 16;138(2):122), colitis, ileitis, and Crohn's disease.

[0340] Examples of autoimmune skin diseases include, but are not limited to, autoimmune bullous skin diseases such as pemphigus vulgaris, bullous pemphigoid, and pemphigus foliaceus.

[0341] Examples of autoimmune liver diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 March;54(3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 November;91(5):551; Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 June;11(6):595), and autoimmune hepatitis (Manns MP. J Hepatol 2000 August;33(2):326).

[0342] Examples of autoimmune neurological disorders include multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1;112(1-2):1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. and Kraig E, Int Rev Immunol 1999;18(1-2):83; Oshima M. et al., Eur J Immunol 1990 Dec;20(12):2563), neuropathy, motor neuropathy (Kornberg AJ. J Clin Neurosci. 2000 May;7(3):191); Guillain-Barre syndrome and autoimmune neuropathy (Kusunoki S., Am J Med Sci. 2000 Apr;319(4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319(4):204); paraneoplastic neurological disorders, cerebellar atrophy, paraneoplastic cerebellar atrophy, and stiff-man syndrome (Hiemstra HS. et al. Proc Natl Acad Sci units SA 2001 Mar 27;98(7):3988); non-paraneoplastic stiff-man syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen encephalitis, amyotrophic lateral sclerosis, Sydenham chorea, Gilles de la Tourette syndrome, and autoimmune polyendocrinopathy (Antoine JC. and Honnorat J. Rev Neurol (Paris) January 2000;156(1):23); immune neuropathy (Nobile-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); Isaacs syndrome, arthrogryposis multiplex congenita (Vincent A. et al., Ann NY Acad Sci. May 13, 1998;841:482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry May 1994;57(5):544), and neurodegenerative diseases.

[0343] Examples of autoimmune muscle diseases include, but are not limited to, myositis, autoimmune myositis, and primary Sjogren's syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123(1):92), and smooth muscle autoimmune disorders (Zauli D. et al., Biomed Pharmacother 1999 Jun;53(5-6):234).

[0344] Examples of autoimmune kidney diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;1(2):140).

[0345] Examples of autoimmune diseases related to reproduction include, but are not limited to, recurrent fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9).

[0346] Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug;157(1):249), and autoimmune diseases of the inner ear (Gloddek B. et al., Ann NY Acad Sci 1997 Dec 29;830:266).

[0347] Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998;17(1-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6(2):156); Chan OT. et al., Immunol Rev 1999 Jun;169:107).

[0348] According to certain embodiments, the disease is a transplant rejection disease.

[0349] Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection, and graft-versus-host disease.

[0350] According to a particular embodiment, the disease is an allergic disease.

[0351] Examples of allergic diseases include, but are not limited to, asthma, rashes, hives, pollen allergies, dust mite allergies, venom allergies, cosmetic allergies, latex allergies, chemical allergies, drug allergies, stinging insect allergies, animal dander allergies, stinging plant allergies, poison ivy allergies, and food allergies.

[0352] As mentioned above, according to certain embodiments, T cells are administered to a subject in combination with a therapeutic composition that is specific for pathological cells (e.g., binds to an antigen that is overexpressed or singly expressed by pathological (e.g., cancerous) cells compared to non-pathological cells) on the one hand, and is capable of binding to the TCR complex on the other hand.

[0353] According to certain embodiments, the therapeutic composition binds to CD3.

[0354] According to certain embodiments, the therapeutic composition binds to the constant region or hinge region of the TCR.

[0355] According to certain embodiments, the therapeutic composition binds to a heterologous tag fused to or attached to the TCR complex, non-limiting examples of such tags are known in the art and described in further detail herein above.

[0356] The administration of the T cells and the therapeutic composition can be by the same route or by separate routes.

[0357] The T cells may be administered prior to, after, or simultaneously with the therapeutic agent.

[0358] Multiple administrations of the T cells and / or therapeutic composition can be administered.

[0359] According to certain embodiments, multiple distinct therapeutic compositions that are specific for diseased cells (e.g., target different antigens) and capable of binding to the TCR complex are provided to the subject.

[0360] Therapeutic compositions capable of binding to pathological cells and TCR complexes are known in the art and include, but are not limited to, antibodies such as bispecific and trispecific antibodies.

[0361] As used herein, the term "antibody" includes intact molecules as well as functional fragments thereof that are capable of binding to an epitope of an antigen.

[0362] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0363] According to certain embodiments, antibody fragments include single chains, Fab, Fab', and F(ab') 2 These include, but are not limited to, fragments, Fd, Fcab, Fv, dsFv, scFvs, diabodies, minibodies, nanobodies, Fab expression libraries, or single domain molecules such as VH and VL that are capable of binding to an epitope of an antigen in an HLA-restricted manner.

[0364] According to certain embodiments, the therapeutic composition is at least a bispecific antibody.

[0365] As used herein, the term "bispecific antibody" refers to an antibody having two different antigen-binding moieties, where a first binding moiety has affinity for the TCR complex and a second binding moiety has affinity for an antigen different from the TCR complex. According to certain embodiments, the bispecific antibody binds to the TCR complex on the one hand and to an antigen expressed by a pathological cell (e.g., a cancer cell) on the other hand.

[0366] Methods for producing bispecific antibodies are known in the art and are disclosed, for example, in U.S. Pat. Nos. 4,474,893, 5,959,084, U.S. Pat. Nos. 7,235,641, 7,183,076, U.S. Publication No. 20080219980, and International Publication Nos. WO2010 / 115589, WO2013150043, and WO2012118903, all of which are incorporated herein in their entireties, and include, for example, chemical crosslinking (Brennan et al., Science 229, 81 (1985); Raso et al., J. Biol. Chern. 272, 27623 (1997)), disulfide exchange, production of hybrid-hybridomas (quadromas), etc., by transcription and translation to produce a single polypeptide chain embodying the bispecific antibody, or by transcription and translation to produce two or more polypeptide chains that can be covalently associated to produce the bispecific antibody. Contemplated bispecific antibodies can also be made entirely by chemical synthesis.

[0367] Thus, according to certain embodiments, a therapeutic antibody comprises at least one arm that binds to an antigen overexpressed or expressed alone by a pathological cell and one arm that comprises an antibody portion that binds to the TCR complex.

[0368] The choice of therapeutic antibody to be used is well within the capabilities of one skilled in the art and depends on the type of disease and the antigens expressed by the pathological cells associated with the pathology.

[0369] According to certain embodiments, the therapeutic composition comprises an anti-CD3 antibody.

[0370] According to certain embodiments, the anti-CD3 antibody portion is of an antibody selected from the group consisting of L2K, TR66, OKT3UCHT1, humanized UHCT1, F6A, SP34, and I2C.

[0371] According to certain embodiments, the anti-CD3 antibody portion is an antibody selected from the group consisting of L2K, TR66, and OKT3.

[0372] According to certain embodiments, the anti-CD3 antibody portion is an antibody selected from the group consisting of L2K, SP34, and UCHT1.

[0373] According to certain embodiments, the therapeutic antibody binds to an antigen expressed by a cancerous cell. Non-limiting examples of such antigens include CD19, EGFR, HER2, MUC-1, CA-125, mesothelin, ROR1, GPC3, PSCA, CD133, CD70, EpCAM, CEA, CAIX, CD171, GD2, Tn-MUC1, EGFRvIII, ADGRE2, CD33, CD123, CCR1, CLEC12A, LILRB2, BCMA, CD138, gp100, and those described in Q He et al. (2019) J Hematol Oncol 12, 99, the contents of which are incorporated herein by reference in their entirety.

[0374] According to some embodiments of the invention, the therapeutic agent is selected from the group consisting of blinatumomab, AMG330, AMG701, orlotamab, AMG420, CC-93269, APVO436, JNJ-63709178, AMG757, MT110, tebentafsp, odronextamab, RG6007, RG6194, RG6232, RG7828, teclistamab, mosunetuzumab, RG7802 civisatamab, cebostamab, glofitamab, and IMMTAC (e.g., RG6290).

[0375] According to certain embodiments, the pathological cells express CD19 and the therapeutic composition comprises blinatumomab.

[0376] According to a particular embodiment, the pathological cells express EpCAM and the therapeutic composition comprises MT110.

[0377] According to a particular embodiment, the pathological cells express CD20 and the therapeutic composition comprises odronextamab.

[0378] According to a particular embodiment, the pathological cells express CD20 and the therapeutic composition comprises mosunetuzumab.

[0379] According to certain embodiments, the pathological cells express HLA-A2-WT1 and the therapeutic composition comprises RG6007.

[0380] According to certain embodiments, the pathological cells express HER2 and the therapeutic composition comprises RG6194.

[0381] In certain embodiments, the pathological cells express TYRP1 and the therapeutic composition comprises RG6232.

[0382] In certain embodiments, the pathological cells express MAGE-A4 and the therapeutic composition comprises RG6290.

[0383] According to certain embodiments, the therapeutic composition comprises an anti-TCR antibody.

[0384] According to certain embodiments, the anti-TCR antibody portion is of an antibody clone selected from the group consisting of IP26, WT31, T10B9, BW242 / 41, 8A3, 3A8, Jovi-1.

[0385] According to certain embodiments, the T cells and therapeutic compositions capable of binding to pathological cells and TCR complexes can be administered to a subject in combination with other established or experimental therapeutic regimens for treating diseases associated with pathological cells (e.g., cancer), including, but not limited to, analgesics, chemotherapeutic agents, radiotherapeutic agents, cytotoxic therapy (conditioning), hormonal therapy, and other therapeutic regimens known in the art (e.g., surgery).

[0386] The therapeutic compositions capable of binding to the T cells disclosed herein and / or pathological cells and TCR complexes disclosed herein can be administered to the subject per se or in a pharmaceutical composition mixed with a suitable carrier or excipient.

[0387] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0388] As used herein, the term "active ingredient" refers to T cells and / or therapeutic compositions capable of binding to pathological cells and the TCR complex responsible for the biological effect.

[0389] Thus, according to certain embodiments, the T cells are the active ingredient in the formulation.

[0390] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound. Adjuvants are included in these phrases.

[0391] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0392] Techniques for formulation and administration of drugs may be found in the latest edition of "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

[0393] Suitable routes of administration may include, for example, oral, rectal, mucosal, particularly nasal, intestinal or parenteral delivery (including intramuscular, intradermal, subcutaneous and intramedullary injections, as well as intrathecal, direct intraventricular, intracardiac injections (e.g., injection into the right or left ventricular cavity, injection into a common coronary artery), intravenous, intraperitoneal, intranasal, or intraocular injections.

[0394] Conventional approaches for drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion); molecular engineering of drugs that seek to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins containing transport peptides with affinity for endothelial cell surface molecules in combination with drugs that cannot themselves cross the BBB); pharmacological strategies designed to increase the lipid solubility of drugs (e.g., conjugation of water-soluble drugs to lipid or cholesterol carriers); and temporary disruption of the integrity of the BBB by hyperosmotic disruption (resulting from injection of mannitol solutions into the carotid artery or use of biologically active agents such as angiotensin peptides). However, each of these strategies has limitations, such as inherent risks associated with invasive surgical procedures, size limitations imposed by limitations inherent to endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and possible risks of brain damage in regions of the brain where the BBB is disrupted, resulting in suboptimal delivery methods.

[0395] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, by injecting the pharmaceutical composition directly into the tissue area of ​​the patient.

[0396] According to a particular embodiment, the T cells of the present invention or a pharmaceutical composition comprising same are administered via an IV route.

[0397] The pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, powdering, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0398] Thus, pharmaceutical compositions for use in accordance with some embodiments of the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers that include excipients and auxiliaries that facilitate processing of the active ingredients into pharma- ceutically usable preparations. Appropriate formulations depend on the route of administration selected.

[0399] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers (e.g., Hanks' solution, Ringer's solution, or physiological salt buffer). For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0400] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients. Pharmacological preparations for oral use can be made using solid excipients, optionally milling the resulting mixture, and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable auxiliary agents, if desired. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose, etc.; and / or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0401] Dragee cores are provided with suitable coatings.For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0402] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers (e.g., glycerol or sorbitol). Push-fit capsules can contain active ingredients mixed with fillers such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, active ingredients can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Additionally, stabilizers can be added. All formulations for oral administration should be in dosages appropriate for the selected route of administration.

[0403] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0404] For administration by nasal inhalation, the active ingredient for use according to some embodiments of the present invention is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in a dispenser can be formulated, which contain a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0405] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, optionally with the addition of preservatives. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents, such as suspending, stabilizing, and / or dispersing agents.

[0406] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as suitable oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. If necessary, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredient, allowing the preparation of highly concentrated solutions.

[0407] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water-based solution, before use.

[0408] Pharmaceutical compositions of some embodiments of the present invention may be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0409] Alternative embodiments include depot formulations, which provide sustained release or an extended duration of activity of the active ingredient in a subject, as is well known in the art.

[0410] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0411] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0412] For any preparation used in the methods of the present invention, the therapeutically effective amount or dose can be initially estimated from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0413] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, cell culture, or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition. (See, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics", Ch.1, p.1).

[0414] Dosage and interval can be adjusted individually to provide a level of active ingredient sufficient to induce or suppress biological effects (minimal effective concentration MEC). MEC varies for each preparation but can be estimated from in vitro data. Dosage required to achieve MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentration.

[0415] Depending on the severity and responsiveness of the condition to be treated, dosage may be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0416] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0417] The compositions of some embodiments of the present invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be contained with a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the agency of the form of the composition or its administration to humans or animals. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. The compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition, as further detailed above.

[0418] According to another aspect of the invention, an article of manufacture is provided that includes packaging material packaging the T cells disclosed herein and a therapeutic composition capable of binding to a pathological cell and a TCR complex.

[0419] According to certain embodiments, the product is identified for the treatment of diseases associated with pathological cells (eg, cancer).

[0420] According to certain embodiments, the T cells and the therapeutic composition are packaged in separate containers.

[0421] According to certain embodiments, the T cells and the therapeutic composition are packaged in a co-formulation.

[0422] As used herein, the term "about" refers to ±10%.

[0423] The terms "comprises," "comprising," "includes," "including," "having" and their conjugations mean "including but not limited to."

[0424] The term "consisting of" means "including and limited to."

[0425] The term "consisting essentially of" means that a composition, method, or structure may include additional compositions, steps, and / or moieties, but only if the additional compositions, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed component, method, or structure.

[0426] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds (including mixtures thereof).

[0427] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0428] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the given range. The phrases "ranging / ranges between" a first designated number and a second designated number, and "ranging / ranges from" a first designated number to a second designated number, are used interchangeably herein and are meant to include the first and second designated numbers and all fractional and integer numbers therebetween.

[0429] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques, and procedures known to or readily developed by practitioners in the chemical, pharmaceutical, biological, biochemical, and medical arts.

[0430] When referring to a particular sequence listing, such reference should also be understood to encompass sequences that substantially correspond to their complementary sequences, including minor sequence variations resulting from, for example, sequencing errors, cloning errors, or other changes resulting in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1 in 100 nucleotides, alternatively less than 1 in 200 nucleotides, alternatively less than 1 in 500 nucleotides, alternatively less than 1 in 1000 nucleotides, alternatively less than 1 in 5000 nucleotides, alternatively less than 1 in 10,000 nucleotides.

[0431] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention, as appropriate. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0432] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0433] Working Example Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting manner.

[0434] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques which are fully explained in the literature.See, e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al. (1989); Current Protocols in Molecular Biology, vols. I-III, Ausubel, R. M. (ed.) (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds.), Genome Analysis: A Laboratory Manual Series, vols. 1-4, Cold Spring Harbor Laboratory. Press, New York (1998); methods described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659, and 5,272,057; Cell Biology: A Laboratory Handbook, Vols. I-III, Cellis, JE ed. (1994); Culture of Animal Cells-A Manual of Basic Technique, Freshney, Wiley-Liss, New York (1994), 3rd Edition: Current Protocols in Immunology, Vols. I-III, Coligan JE ed. (1994); Stites et al. (eds.), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, Calif. (1994); Mishell and Shiigi (eds.), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).Available immunoassays have been described extensively in the patent and scientific literature, e.g., in U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771; and 5,281,521; "Oligonucleotide Immunoassays," which are incorporated herein by reference in their entirety. See, for example, "Nucleic Acid Hybridization", edited by Gait, MJ (1984); "Transcription and Translation", edited by Hames, BD and Higgins SJ (1985); "Animal Cell Culture", edited by Freshney, RI (1986); "Immobilized Cells and Enzymes", IRL Press (1986); "A Practical Guide to Molecular Cloning", Perbal, B. (1984) and "Methods in Enzymology", volumes 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual", CSHL Press (1996). All of these are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated by reference herein.

[0435] Materials and Methods Cell Culture and Reagents - K562, Raji, and 293T cell lines (JCRB) were cultured as recommended by the supplier. All cell lines were authenticated by short tandem repeat (STR) profiling using the PowerPlex16 HS kit (Promega). Cell number and viability were estimated using a hemocytometer and trypan blue exclusion assay, respectively. Research grade blinatumomab and MT110 were purchased from Oak BioSciences, Inc. (California, USA).

[0436] Generation of endogenous TCR negative T cells - Human peripheral blood mononuclear cells (PBMC) cells were purified from peripheral blood samples of healthy donors using Ficoll-Hypaque density gradient centrifugation. Extracted PBMCs were activated and expanded in 24-well suspension plates in 4Cell® Nutri-T Media (Sartorius, 05-11F2001-1K) supplemented with 500U / mL IL-2 (Peprotech, 200-02) and antibiotics using OKT3 (Biolegend, Cat. No. 317301). 72 hours after activation, cells were electroporated with SpCas9 RNP using the Alt-R CRISPR-Cas9 system and sgRNA targeting human constant alpha region (SEQ ID NO: 1) or human constant beta region (SEQ ID NO: 45) (IDT, Coralville) using AMAXA Nucleofector (Lonza). Electroporated cells were cultured in 4Cell® Nutri-T medium supplemented with 500 U / mL IL-2 and antibiotics. Three days after electroporation, most cells were negative (>85%) for stationary α or β chains as determined by anti-CD3 flow antibody (PE-Cy7OKT3 Biolegend, Cat. No. 317333). Negative cells were further enriched by magnetic anti-CD3 beads (Miltenyi Biotec, Cat. No. 130-050-101) according to the manufacturer's instructions to reach 95%-99.9% CD3- / TCR cells (Figure 3A). To knock out both α and β chains, cells were first electroporated with gRNA targeting the α chain (SEQ ID NO: 1), negatively selected by magnetic beads, and then further electroporated with sgRNA targeting the β region (SEQ ID NO: 45), followed by magnetic bead purification of CD3- / TCR- T cells.

[0437] DNA constructs and cloning - RTV-020 retroviral vector (Cell Biolabs Inc., San Diego, CA) was used. For each construct, the amino acid sequence was optimized (IDT, Coralville) and each sequence was designed to express a different TCR α and / or β polypeptide, a V5-Furin-P2A sequence (SYang et al., 2008) and a polypeptide encoding EGFP or truncated NGFR (ΔNGFR) as indicated. Schematic diagrams of the designed constructs are shown in Figures 2A-B, with amino acid sequences provided in SEQ ID NOs: 2-3, 26-32, 46, 48-52, 57, 59, 61-69, 109, 123, 125, and 127, and nucleic acid sequences provided in SEQ ID NOs: 33-41, 53-56, 58, 60, 70-80, 110, 124, 126, and 128. The final sequences were ordered as gBlocks® (IDT, Coralville). gBlocks® were amplified using appropriate primers and High fidelity PrimeSTAR GXL DNA polymerase (catalog number R050B, TAKARA), then digested with BamHI-HF and XhoI (NEB) and ligated into the retroviral vector. Where indicated, three mutations were introduced into the transmembrane domain of the α chain: S116L, G119V, and F120L (denoted as LVL). Where indicated, cysteines were introduced into both the α and β chains (Cys): T48C in the α chain and S57C in the β chain, L12C in the α chain and S17C in the β chain, Y43C in the α chain and L63C in the β chain, S61C in the α chain and R79C in the β chain, L12C in the α chain and F14C in the β chain, V22C in the α chain and F14C in the β chain, Y10C in the α chain and S17C in the β chain, T45C in the α chain and D59C in the β chain, L50C in the α chain and S57C in the β chain, S61C in the α chain and S57C in the β chain, T45C in the α chain and S77C in the β chain, S15C in the α chain and V13C in the β chain, or S15C in the α chain and E15C in the β chain. Where indicated (marked mm), several mutations were introduced into the extracellular domains of the α and β chains as follows: α chain mutations P91S, E92D, S93V, S94P; β chain mutations: E18K, S22A, F133I, E / V136A, Q139H.Where indicated (denoted as Des), several mutations were introduced into the α and β chains as follows: α chain mutations S21F, T32I, A72T and β chain mutations E18K, H23R, D39P, S54D. Sanger sequencing was used to verify the cloned vectors. Similarly, Ba9 (SEQ ID NO: 83-84) and α6 (SEQ ID NO: 81-82) constructs disclosed by International Patent Application Publication No. WO2020138371 were designed and cloned.

[0438] Virus preparation and transduction procedure - Viral particles were generated by transient transfection of 293T cells with contracted retroviral vectors using CMV-gagPol and CMV-VSVG (Cell Biolabs, Inc.). CRISPR-edited TCR-negative T cells were seeded on tissue culture plates coated with Retronectin (TaKaRa Bio) and concentrated viral particles were added. Cells were incubated with the virus preparation for 7 hours, and then the medium was replaced with 4Cell® Nutri-T medium supplemented with 500 U / mL IL-2 and antibiotics.

[0439] Flow cytometry analysis - Cells were stained with OKT3 anti-CD3-APC (Biolegend) according to the manufacturer's instructions. Results were obtained using Gallios™ and Cytoflex Flow Cytometers (Beckman Coulter, Inc.). Flow cytometry results were analyzed using FlowJo v10 software.

[0440] In vitro antitumor effect - Endogenous TCR negative T cells were infected with BluT containing T48C and LVL mutations and a truncated β chain containing S57C mutation (SEQ ID NO: 125-126) and selected with magnetic beads using the ΔNGFR marker. Cells were then incubated with Raji-F.Luc CD19+ lymphoma cells (Cat. No. IFO50046JCRB) at various E:T ratios in the presence or absence of 500 μg / mL Blinatumomab (CD19 BiTE) at 37°C for 24 hours, and firefly luciferase activity was measured by GloMax® Discover Microplate Reader to determine cytotoxicity. Anti-CD19 CAR-T cells were prepared by infecting endogenous TCR-negative T cells with anti-CD19(FMC63)-CD28z (sequence numbers 127-128) and selecting with magnetic beads using the ΔNGFR marker, and non-modified CD3-positive TCR-positive T cells were used as a positive control.

[0441] In vivo antitumor effect - 2 × 10 6 Raji-F.Luc, 10 7 T cells (endogenous TCR-negative T cells infected with vectors encoding a truncated α-chain containing the T48C and LVL mutations and a truncated β-chain containing the S57C (SEQ ID NOs: 125-126) and selected with magnetic beads using the ΔNGFR marker) (n=10); endogenous TCR-negative anti-CD19 CAR-T cells infected with anti-CD19(FMC63)-CD28z and selected with magnetic beads using the ΔNGFR marker (n=8); or unmodified CD3+TCR+ T cells (n=16) were subcutaneously injected into NSG mice with (n=34) or without (n=8). 24 hours after injection, the indicated groups were treated intravenously with 5 μg / mouse of blinatomumab for 5 consecutive days. Bioluminescence imaging (IVIS® Lumina X5, PerkElmer) was performed weekly until day 60 to follow tumor growth.

[0442] Example 1 Generation of truncated T cell receptors (TCRs) We have devised a novel engineered TCR lacking the variable regions of the TCR α and β chains. This TCR, referred to herein as "blunt truncated TCR (BluT)", is presented as part of a TCR complex on the cell surface (Figure 1A) and can function as a TCR that does not recognize any antigen via the MHC mechanism, thus preventing the induction of GvHD even in the case of mismatched pairing with endogenous TCR α or β chains (Figure 1B).

[0443] To this end, several constructs encoding human TCR α-chains and / or β-chains lacking their variable regions were designed (Figures 2A-B, amino acid sequences are provided in SEQ ID NOs: 2-3 and 26-32, 46-52, 57, 59, 61-69, 81, 83, 109, 123, 125, and 127, and nucleic acid sequences are provided in SEQ ID NOs: 33-41, 53-56, 58, 60, 70, 80, 82, 84, 124, 126, and 128), packaged into retroviruses, and used to infect TCR-negative T cells (formed by CRISPR targeting of the TCR α-chain) (Figures 3A-B). EGFP was used as a marker of stable infection. As shown in Figures 3B and 4A and Table 1 below, introduction of vectors encoding only truncated α-chains or only truncated β-chains did not result in the presentation of TCR complexes on the cell surface (manifested by the absence of expression of CD3 on the surface). Furthermore, introduction of vectors encoding truncated α and β chains (either the TRBC1 or TRBC2 chains) did not result in the presentation of TCR complexes on the cell surface.

[0444] To address this issue, the inventors explored modifications that would stabilize truncated TCRs (Figures 3B-8B and Table 1 below).

[0445] Introduction of vectors encoding truncated α-chains with stabilizing mutations S116L, G119V, and F120L (denoted as LVL) and truncated β-chains; or vectors encoding truncated α-chains and β-chains with computationally designed stabilizing mutations, i.e., S21F, T32I, A72T in the α-chain and E18K, H23R, D39P, S54D in the β-chain (denoted as Des), did not result in the presentation of TCR complexes on the cell surface (Figures 4 and 7). Furthermore, attempts to express in primary T cells that do not express endogenous α-chains, as disclosed in International Patent Application Publication No. WO2020138371, did not result in CD3 re-expression, either alone or with a complete constant β-chain (Figure 8B).

[0446] However, introduction of vectors encoding a truncated α chain containing the T48C mutation and a truncated β chain containing the S57C mutation, resulting in the formation of an additional disulfide bond between the α and β chains, resulted in the presentation of the TCR complex on the cell surface (as evidenced by the re-expression of CD3 on the surface) (Figures 3B and 4). Furthermore, the addition of LVL mutations to the T48 / S57 cysteine ​​mutations improved CD3 re-expression on the cell surface (Figure 4). Importantly, the introduction of mutations resulting in additional disulfide bonds between the α and β chains at other applicable cysteine ​​sites (SEQ ID NOs: 85-108; see, e.g., Boulter et al., 2003), did not allow CD3 re-expression (Figure 6). Furthermore, removal of 11 or more amino acids from the N-terminus of the constant domain of the α chain was found to destabilize the complex and prevent CD3 expression (Figure 8A).

[0447] In addition, introduction of vectors encoding truncated α- and β-chains containing minimal mouse amino acid substitutions (designated as mm), i.e., P91S, E92D, S93V, S94P in the α-chain and E18K, S22A, F133I, E / V136A, Q139H in the β-chain, also resulted in the presentation of TCR complexes on the cell surface, as evidenced by re-expression of CD3 on the surface (Figure 7). Similar to the cysteine ​​mutations, the addition of the LVL modification to the mm modification further enhanced CD3 re-expression.

[0448] To show that these results are not exclusively valid when knocking out the endogenous α chain to prevent cell surface expression of endogenous TCR, SpCas9 RNP was used to target the endogenous β chain (Figure 5). Similarly, introduction of vectors encoding truncated α and β chains did not result in the presentation of TCR complexes on the cell surface, but introduction of vectors encoding truncated α chains containing the T48C mutation and truncated β chains containing the S57C mutation resulted in the presentation of TCR complexes on the cell surface (as revealed by re-expression of CD3 on the surface). Knocking out both the α and β endogenous chains had no further effect.

[0449] In summary, in order to express a TCR complex containing human TCR α and β chains lacking variable domains on the surface of T cells, modifications to the human TCR wild type sequence must be introduced. The following possible and sufficient modifications were identified: replacement of T48 of the α chain and S57 of the β chain with cysteine ​​residues and / or replacement of some residues in both chains with their mouse counterparts. The additional introduction of LVL mutations in the α chain improves cell surface expression.

[0450] [Table 1]

[0451] Example 2 T cells expressing BluTTCR in combination with a bispecific T cell engager induce efficient in vitro tumor cell lysis. Since the therapeutic strategy of some embodiments is based on injecting T cells expressing BluT into a subject together with a bispecific antibody that binds the TCR complex (e.g., CD3) on the one hand and a cancer antigen on the other hand, the cytotoxic activity of the transduced T cells can be evaluated in vitro, for example on CD19+Raji cells and / or EGFR+K562 cells, in combination with, for example, the anti-CD19 bispecific T cell engager (BiTE) Blinatumomab and / or the anti-EPCAM BiTE-MT110. T cell activation can be determined, for example, by expression of CD107 and secretion of cytokines such as IFNγ, TNFα, IL-2. Cytotoxic activity can be determined, for example, by change in RLU, reduction of CD19 or EGFR percentage compared to untreated controls. For positive selection of transduced T cells by BluT, ΔNGFR can be used as a selection marker (instead of EGFP as described in Figures 2A-B and Example 1 above).

[0452] To this end, endogenous TCR-negative T cells expressing a truncated α-chain containing the T48C and LVL mutations and a truncated β-chain containing S57C were incubated with the Raji-F.Luc CD19-positive lymphoma line at various E:T ratios in the presence or absence of blinatumomab (CD19 BiTE) for 24 hours and cytotoxicity was determined by measuring firefly luciferase activity (Figure 9). Endogenous TCR-negative anti-CD19 CAR-T cells and unmodified CD3-positive TCR-positive T cells were used as positive controls.

[0453] Unlike unmodified T cells containing an intact endogenous TCR (which can recognize tumor cells via the MHC-TCR mechanism and thus have cytotoxic activity against target cells without the addition of BiTEs), T cells containing engineered truncated TCRs have anti-tumor activity only in the presence of anti-CD3 mediators. Moreover, T cells containing engineered truncated TCRs were significantly more cytotoxic compared to unmodified T cells or anti-CD19 CAR-T cells.

[0454] Example 3 T cells expressing the BluT TCR in combination with a bispecific T cell engager induce effective in vivo anti-tumor efficacy. Since the therapeutic strategy of some embodiments is based on injecting BluT-expressing T cells together with a bispecific antibody that binds the TCR complex (e.g., CD3) on the one hand and a cancer antigen on the other hand into a subject, the cytotoxic activity of endogenous TCR-negative T cells expressing a truncated α-chain containing the T48C and LVL mutations and a truncated β-chain containing S57C was evaluated in vivo in mice transplanted with CD19+Raji cells in combination with the anti-CD19 BiTE Blinatumomab. Endogenous TCR-negative anti-CD19 CAR-T cells and unmodified CD3+TCR-positive T cells were used as positive controls.

[0455] As shown in FIG. 10, T cells containing engineered truncated TCRs have anti-tumor activity only in the presence of anti-CD3 mediators.

[0456] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0457] It is the intention of the applicant that all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference at the time of reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application are incorporated herein by reference in their entirety.

[0458] References (Other references are cited throughout this application). 1. MacKay M, Afshinnekoo E, Rub J, Hassan C, Khunte M, Baskaran N et al. The therapeutic landscape for cells engineered with chimeric antigen receptors. Nat Biotechnol [Internet]. 2020 [Cited 20 January 2020]; Available at: www(dot)ncbi(dot)nlm(dot)nih(dot)gov / pubmed / 31907405. 2. Sadelain M, Riddell S. Therapeutic T cell engineering. Nature [Internet]. Nature Publishing Group; 2017; 545: 423-31. Available at: dx(dot)doi(dot)org / 10(dot)1038 / nature22395. 3. Fesnak AD, June CH, Levine BL. Engineered T cells: The promise and challenges of cancer immunotherapy. Nat Rev Cancer [Internet]. Nature Publishing Group; 2016; 16:566-81. Available at: dx(dot)doi(dot)org / 10(dot)1038 / nrc(dot)2016(dot)97. 4. Kershaw MH, Westwood JA, Darcy PK. Gene-engineered T cells for cancer therapy. Nat Rev Cancer [Internet]. Nature Publishing Group; 2013; 13:525-41. Available at: dx(dot)doi(dot)org / 10(dot)1038 / nrc3565. [ PubMed ] [ Cross Ref ] 5.Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F, Khoshtinat Nikkhoi S. Front.Oncol.Frontiers Media SA;2021[May 27, 2021] 924 months at www(dot)frontiersin(dot)org. [ PubMed ] [ Cross Ref ] 6.Goebeler ME、Bargou RC.T cell-engaging therapies-BiTEs and beyond.Nat Rev Clin Oncol[Medical]Springer US;2020;17:418-34 dx(dot)doi(dot)org / 10(dot)1038 / s41571-020-0347-5. 7.Rajat Bannerji,MD,PhD,John N.Allan,MD,John E.Arnason,MD,Jennifer R.Brown,MD,PhD,Ranjana Advani,MD,Stephen M.Ansell,MD,PhD,Susan M.O'Brien,MD,Johannes Duell,MD,Peter Martin,FRCPC,MD,MS,Robin M.Joyce,MD,Jingjin Li,PhD,Dina MMOdronextamab(REGN1979),a Human CD20 x CD3 Bispecific Antibody,Induces Durable,Complete Responses in Patients with Highly Refractory B-Cell Non-Hodgkin Lymphoma,Including Patients Refractory to CAR T Therapy.2020 at ash(dot)confex(dot)com / ash / 2020 / webprogram / Paper136659(dot)html. 8. Ahamadi-Fesharaki R, Fateh A, Vaziri F, Solgi G, Siadat SD, Mahboudi F, et al. Single-Chain Variable Fragment-Based Bispecific Antibodies: Hitting Two Targets with One Sophisticated Arrow.Mol Ther-Oncolytics[Internet].Elsevier Ltd.2019;14:38-56. Available from doi(dot)org / 10(dot)1016 / j(dot)omto(dot)2019(dot)02(dot)004. 9.Garber K.Driving T-cell immunotherapy to solid tumors.Nat Biotechnol.Nature Publishing Group;2018;36:215-9. 10. Slaney CY, Wang P, Darcy PK, Kershaw MH. CARs versus biTEs: A comparison between T cell-redirection strategies for cancer treatment [Internet]. Cancer Discov. American Association for Cancer Research Inc.; 2018 [cited 2021-05-27]. pp. 924-34. Available from: www(dot)aacrjournals(dot)org. 11.Chandran SS, Klebanoff CA. T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance. Immunol Rev. 2019;290:127-47. 12. Gudipati V, Rydzek J, Doel-Perez I, Goncalves VDR, Scharf L, Koenigsberger S, et al. Inefficient CAR-proximal signaling blunts antigen sensitivity. Nat Immunol.2020;21:848-56. 13. Helsen CW, Hammill JA, Lau VWC, Mwawasi KA, Afsahi A, Bezverbnaya K, et al. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity.Nat Commun[Internet].Springer US;2018;9:1-13. Available from dx(dot)doi(dot)org / 10(dot)1038 / s41467-018-05395-y. 14. Baeuerle PA, Ding J, Patel E, Thorausch N, Horton H, Gierut J, et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nat Commun [Internet]. Springer US; 2019; 10: 1-12. Available from: dx(dot)doi(dot)org / 10(dot)1038 / s41467-019-10097-0. 15. Lee YG, Chu H, Lu Y, Leamon CP, Srinivasarao M, Putt KS, et al. Regulation of CAR T cell-mediated cytokine release syndrome-like toxicity using low molecular weight adapters. Nat Commun [Internet]. Springer US; 2019;10:1-11. Available from: dx.doi.org / 10.1038 / s41467-019-10565-7. 16. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat. Med. Nature Publishing Group; 2019. 1408-14 pages. 17. Benjamin R, Graham C, Yallop D, Jozwik A, Mirci-Danicar OC, Lucchini G, et al. Genome-edited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies. Lancet. 2020;396:1885-94. 18. Alderton GK. Immunotherapy: Engineered T cells for all. Nat. Rev. Cancer. Nature Publishing Group; 2017, 206-7 pages. 19. Van Loenen MM, De Boer R, Amir AL, Hagedoorn RS, Volbeda GL, Willemze R, et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci USA. 2010;107:10972-7. 20. Mack M, Gruber R, Schmidt S, Riethmuller G, Kufer P. Biologic properties of a bispecific single-chain antibody directed against 17-1A (EpCAM) and CD3: tumor cell-dependent T cell stimulation and cytotoxic activity. J Immunol. 1997;158. 21. Exley M, Terhorst C, Wileman T. Structure, assembly and intracellular transport of the T cell receptor for antigen. Semin Immunol. 1991;3:283-97. 22. Garfall AL, Dancy EK, Cohen AD, Hwang WT, Fraietta JA, Davis MM, et al. T-cell phenotypes associated with effective CAR T-cell therapy in postinduction vs relapsed multiple myeloma. Blood Adv. 2019;3:2812-5. 23. Lamers CHJ, Sleijfer S, Vulto AG, Kruit WHJ, Kliffen M, Debets R, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol [Internet]. 2006 [cited 20 January 2020]; 24:e20-2. Available from: www.ncbi.nlm.nih.gov / pubmed / 16648493. 24. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of t cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010;18:843-51. 25. Linette GP, Stadtmauer EA, Maus MV., Rapoport AP, Levine BL, Emery L, et al. Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood. 2013;122:863-71. 26. Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J.Immunother. 2013;36:133-51. 27. Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells: Recognition and management. Blood. American Society of Hematology; 2016; pp. 3321-30. 28. Maude SL, Barrett D, Teachey DT, Grupp SA. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. (USA). Lippincott Williams and Wilkins;2014.pp.119-22. 29. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric Antigen Receptor-Modified T Cells for Acute Lymphoid Leukemia.N Engl J Med [Internet].2013 [cited January 20, 2020];368:1509-18. Available from www(dot)nejm(dot)org / doi / 10(dot)1056 / NEJMoa1215134. 30. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.Nat Med.Nature Publishing Group;2018;24:20-8. 31. Choi BD, Yu X, Castano AP, Bouffard AA, Schmidts A, Larson RC, et al. CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat Biotechnol. Nature Publishing Group;2019;37:1049-58. 32. Liu Y, Liu G, Wang J, Zheng ZY, Jia L, Rui W, et al. Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors.Sci Transl Med.2021;13. 33. Yang S, Cohen CJ, Peng PD, Zhao Y, Cassard L, Yu Z et al. Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther[Internet]. Nature Publishing Group; 2008 [cited 28 May 2021]; 15:1411-23. Available at: www(dot)nature(dot)com / gt. 34. Haga-Friedman A, Horovitz-Fried M, Cohen CJ. Incorporation of Transmembrane Hydrophobic Mutations in the TCR Enhance Its Surface Expression and T Cell Functional Avidity.J Immunol.2012;188:5538-46. 35. Jin BY, Campbell TE, Draper LM, Stevanovic S, Weissbrich B, Yu Z, et al. Engineered T cells targeting E7 mediate regression of human papillomavirus cancers in a murine model. JCI insight. 2018;3. 36. Kuball J, Hauptrock B, Malina V, Antunes E, Voss RH, Wolfl M, et al. Increasing functional avidity of TCR-Redirected T cells by removing defined N-glycosylation sites in the TCR constant domain. J Exp Med. 2009;206:463-75. 37. Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, Morgan RA. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res. 2007;67:3898-903. 38. Boulter JM, Glick M, Todorov PT, Baston E, Sami M, Rizkallah P, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng. 2003;16:707-11. 39. Kuball J, Dossett ML, Wolfl M, Ho WY, Voss RH, Fowler C, et al. Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood. 2007;109:2331-8. 40. Froning K, Maguire J, Sereno A, Huang F, Chang S, Weichert K et al. Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics. Nat Commun [Internet]. Springer US;2020;11:1-14. Available at: dx(dot)doi(dot)org / 10(dot)1038 / s41467-020-16231-7. 41. Sommermeyer D, Uckert W. Minimal Amino Acid Exchange in Human TCR Constant Regions Fosters Improved Function of TCR Gene-Modified T Cells.J Immunol.2010;184:6223-31. 42. Brischwein K, Schlereth B, Guller B, Steiger C, Wolf A, Lutterbuese R, et al. MT110: A novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Mol Immunol [Internet]. Mol Immunol; 2006 [cited 30 May 2021]; 43: 1129-43. Available from: pubmed(dot)ncbi(dot)nlm(dot)nih(dot)gov / 16139892 / .

Claims

**Claim 1** A T cell receptor (TCR) comprising a human TCRα polypeptide and a human TCRβ polypeptide, wherein the TCR lacks an antigen-binding domain and a heterologous extracellular binding domain, and the TCRα and β polypeptides comprise amino acid modifications that enable presentation of the TCR as a TCR complex on the surface of T cells that express the TCR, said modifications being (i) comprising a T48C amino acid substitution corresponding to the human TCRα polypeptide set forth in SEQ ID NO: 9 and an S57C amino acid substitution corresponding to the human TCRβ polypeptide set forth in SEQ ID NO: 12, and / or (ii) a TCR wherein the TCRα polypeptide and the TCRβ polypeptide comprise chimeric human and mouse TCRα polypeptides and chimeric human and mouse TCRβ polypeptides. **Claim 2** A T cell receptor (TCR) comprising a human TCRα polypeptide and a human TCRβ polypeptide, wherein the TCR lacks an antigen-binding domain, and the TCRα and β polypeptides comprise amino acid modifications that enable presentation of the TCR as a TCR complex on the surface of T cells that express the TCR, said modifications being (i) a T48C, S116L, G119V, and F120L amino acid substitution corresponding to the human TCRα polypeptide set forth in SEQ ID NO: 9, and an S57C mutation corresponding to the human TCRβ polypeptide set forth in SEQ ID NO: 12, and / or (ii) a P91S, E92D, S93V, and S94P amino acid substitution corresponding to the human TCRα polypeptide set forth in SEQ ID NO: 9, and an E18K, S22A, F133I, E / V136A, and Q139H amino acid substitution corresponding to the human TCRβ polypeptide set forth in SEQ ID NO: 12, comprising a TCR. **Claim 3** The TCR according to claim 2, wherein the modification of (ii) further comprises an S116L, G119V, and F120L amino acid substitution corresponding to the human TCRα polypeptide set forth in SEQ ID NO:

9. **Claim 4** The TCR according to claim 1, wherein the modification further comprises an S116L, G119V, and F120L amino acid substitution corresponding to the human TCRα polypeptide set forth in SEQ ID NO:

9. **Claim 5** The TCR according to claim 1 or 4, wherein the modification in (ii) includes the P91S, E92D, S93V, and S94P amino acid substitutions corresponding to the human TCRα polypeptide set forth in SEQ ID NO: 9, and the E18K, S22A, F133I, E / V136A, and Q139H amino acid substitutions corresponding to the human TCRβ polypeptide set forth in SEQ ID NO:

12.

6. The TCR according to claim 1, wherein the TCRα polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 20-23, 47, 111, 114, and 117-120.

7. The TCR according to claim 1 or 2, wherein the TCRα polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 20-23, 47, 111, and 117-120.

8. The TCR according to any one of claims 1-2 and 6, wherein the TCRβ polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-16, 18-19, 24-25, 112-113, 115-116, and 121-122.

9. At least one polynucleotide encoding the TCR according to any one of claims 1-2 and 6.

10. A transduced cell that expresses the TCR according to any one of claims 1-2 and 6.

11. A transduced cell that expresses at least one polynucleotide according to claim 9.

12. A transduced T cell that expresses the TCR according to any one of claims 1-2 and 6.

13. A transduced T cell that expresses at least one polynucleotide according to claim 9.

14. A method for producing a TCR-expressing cell, the method comprising introducing at least one polynucleotide according to claim 9 into a cell under conditions that allow expression of the TCR.

15. The cell according to claim 12, wherein the cell does not express an endogenous TCR.

16. The cell according to claim 13, wherein the cell does not express an endogenous TCR.

17. The method according to claim 14, wherein the cell does not express an endogenous TCR.

18. The method according to claim 15, further comprising downregulating the expression of the endogenous TCR.

19. A therapeutic composition for use in treating a disease associated with diseased cells in a subject in need thereof, the T cells according to claim 12, and capable of binding to the diseased cells and a TCR complex comprising the TCR.

20. A therapeutic composition for use in treating a disease associated with diseased cells in a subject in need thereof, the T cells according to claim 15, and capable of binding to the diseased cells and a TCR complex comprising the TCR.

21. A product comprising a packaging material for packaging a cell according to claim 10 and a therapeutic composition capable of binding to a diseased cell and a TCR complex comprising the TCR.

22. A product comprising a packaging material for packaging a cell according to claim 12 and a therapeutic composition capable of binding to a diseased cell and a TCR complex comprising the TCR.

23. A product comprising a packaging material for packaging a cell according to claim 15 and a therapeutic composition capable of binding to a diseased cell and a TCR complex comprising the TCR.

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