WO2021019472A1 - T cell receptors and methods of use thereof - Google Patents

T cell receptors and methods of use thereof Download PDF

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Publication number
WO2021019472A1
WO2021019472A1 PCT/IB2020/057172 IB2020057172W WO2021019472A1 WO 2021019472 A1 WO2021019472 A1 WO 2021019472A1 IB 2020057172 W IB2020057172 W IB 2020057172W WO 2021019472 A1 WO2021019472 A1 WO 2021019472A1
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Prior art keywords
drb1
dpb1
dqb1
tcr
amino acid
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PCT/IB2020/057172
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English (en)
French (fr)
Inventor
Naoto Hirano
Kenji SUGATA
Kayoko SASO
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University Health Network
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University Health Network
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Priority to CN202080061077.3A priority Critical patent/CN114555815B/zh
Priority to US17/631,825 priority patent/US12570715B2/en
Priority to MX2022001209A priority patent/MX2022001209A/es
Priority to BR112022001688A priority patent/BR112022001688A2/pt
Priority to CA3146298A priority patent/CA3146298A1/en
Priority to JP2022506470A priority patent/JP7669346B2/ja
Application filed by University Health Network filed Critical University Health Network
Priority to EP20845977.6A priority patent/EP4004217A4/en
Priority to AU2020320445A priority patent/AU2020320445A1/en
Priority to KR1020227006433A priority patent/KR20220087431A/ko
Publication of WO2021019472A1 publication Critical patent/WO2021019472A1/en
Anticipated expiration legal-status Critical
Priority to IL290227A priority patent/IL290227A/en
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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Definitions

  • TCRs T cell receptors
  • CCNDl human Gl/S-specific cyclin-Dl
  • T cell therapies are at the forefront of immunotherapeutic development, and adoptive transfer of antitumor T cells has been shown to induce clinical responses in cancer patients. Though many T cell therapies target mutated tumor antigens, the vast majority of neoantigens are not shared and are unique to each patient.
  • non-mutated antigens outnumber mutated antigens by multiple orders of magnitude.
  • the elucidation of T cell epitopes derived from shared antigens may facilitate the robust development of efficacious and safe adoptive T cell therapies that are readily available to a larger cohort of cancer patients.
  • the sheer number of non-mutated antigens and the high polymorphism of HLA genes may have hampered comprehensive analyses of the specificity of antitumor T cell responses toward non-mutated antigens.
  • Certain aspects of the present disclosure are directed to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds human Gl/S-specific cyclin-Dl (CCND1) ("anti-CCNDl TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR, wherein the anti-CCNDl TCR cross competes for binding to human CCND1 with a reference TCR, which comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • Certain aspects of the present disclosure are directed to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds human CCNDl ("anti-CCNDl TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR, wherein the anti-CCNDl TCR binds the same epitope or an overlapping epitope of human CCNDl as a reference TCR, which comprises an alpha chain and a beta chain, wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR binds to an epitope of CCNDl consisting of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the epitope is complexed with an HLA class P molecule.
  • the HLA class P molecule is an HLA-DP, HLA-DQ, or HLA-DR allele, or any combination thereof.
  • the HLA class P molecule is an HLA-DP allele.
  • the HLA class P molecule is an HLA-DP4 allele.
  • the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain comprises variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; wherein the alpha chain CDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7.
  • the beta chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain comprises variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; wherein the beta chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10.
  • the alpha chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 7.
  • the alpha chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 5.
  • the beta chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 8.
  • the alpha chain CDR2 of the anti- CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 6.
  • the beta chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 9.
  • the alpha chain variable domain of the anti-CCNDl TCR comprises an amino acid sequence of a variable domain present in the amino acid sequence set forth SEQ ID NO: 1.
  • the beta chain variable domain of the anti-CCNDl TCR comprises an amino acid sequence of a variable domain present in the amino acid sequence set forth SEQ ID NO: 2.
  • the alpha chain of the anti-CCNDl TCR further comprises a constant region, wherein the constant region is different from endogenous constant region of the alpha chain.
  • the alpha chain of the anti-CCNDl TCR further comprises a constant region, wherein the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth SEQ ID NO: 1.
  • the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth SEQ ID NO: 1.
  • the beta chain of the anti-CCNDl TCR further comprises a constant region, wherein the constant region is different from endogenous constant regions of the beta chain.
  • the beta chain of the anti-CCNDl TCR further comprises a constant region, wherein the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth SEQ ID NO: 2.
  • the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth SEQ ID NO: 2.
  • the alpha chain of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 1.
  • the beta chain of the anti- CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • the second nucleotide sequence is one or more siRNAs that reduce the expression of endogenous TCRs.
  • the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCRs.
  • the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 53-56.
  • the anti-CCNDl TCR comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within the target sequence relative to the corresponding amino acid sequence of an endogenous TCR.
  • the alpha chain comprises a signal peptide
  • the beta chain comprises a signal peptide
  • both the alpha chain and the beta chain comprise a single peptide.
  • the signal peptide comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 20-22 and any combination thereof.
  • the vector is a viral vector, a mammalian vector, or bacterial vector.
  • the vector is a retroviral vector.
  • the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, a hybrid vector, and an adeno associated virus (AAV) vector.
  • the vector is a lentivirus.
  • TCR T cell receptor
  • an antigen binding portion thereof comprising the alpha chain variable domain of an anti- CCNDl TCR disclosed herein and the beta chain variable domain of an anti-CCNDl TCR disclosed herein.
  • Certain aspects of the present disclosure are directed to a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds human CCND1 ("an anti-CCNDl TCR"), which cross competes for binding to human CCND1 with a reference TCR; wherein the reference TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; and wherein the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein the alpha chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1 or the beta chain constant region comprises an amino acid sequence having a least 1, at least 2, at
  • Certain aspects of the present disclosure are directed to a recombinant T cell receptor (TCR) or an antigen binding portion thereof that specifically binds human CCND1 ("an anti-CCNDl TCR"), which binds the same epitope or an overlapping epitope of human CCND1 as a reference TCR; wherein the reference TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2; and wherein the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein the alpha chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1 or the beta chain constant region comprises an amino acid
  • the anti-CCNDl TCR binds to an epitope of CCNDl consisting of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the epitope is complexed with an HLA class P molecule.
  • the HLA class P molecule is an HLA-DP, HLA-DQ, or HLA-DR allele, or any combination thereof.
  • the HLA class P molecule is an HLA-DP allele.
  • the HLA class P molecule is selected from an HLA-DP4 allele.
  • the alpha chain of the anti-CCNDl TCR comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain of the anti-CCNDl TCR comprises variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; wherein the alpha chain CDR3 of the anti-CCNDl comprises an amino acid sequence as set forth in SEQ ID NO: 7.
  • the beta chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10.
  • the alpha chain of the anti-CCNDl TCR comprises a variable domain comprising an alpha chainCDRl, an alpha chain CDR2, and an alpha chain CDR3; wherein the beta chain of the anti-CCNDl TCR comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; and wherein the beta chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 10. In some aspects, the alpha chain CDR3 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 7.
  • the alpha chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 5.
  • the beta chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 8.
  • the alpha chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 6.
  • the beta chain CDR2 of the anti- CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 9.
  • the alpha chain variable domain of the anti-CCNDl TCR comprises an amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 1.
  • the beta chain variable domain of the anti- CCNDl TCR comprises an amino acid sequence of a variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.
  • the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of a constant region present in the amino acid sequence set forth in SEQ ID NO: 1.
  • the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of a constant region present in the amino acid sequence set forth in SEQ ID NO: 2.
  • the alpha chain of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 1.
  • the beta chain of the anti- CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • the alpha chain comprises a signal peptide
  • the beta chain comprises a signal peptide
  • both the alpha chain and the beta chain comprise a single peptide.
  • the signal peptide comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 20-22 and any combination thereof.
  • Certain aspects of the present disclosure are directed to a bispecific TCR comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a TCR or an antigen-binding portion thereof disclosed herein.
  • the first antigen-binding domain comprises a single chain variable fragment ("scFv").
  • the second antigen-binding domain binds specifically to a protein expressed on the surface of a T cell.
  • the second antigen-binding domain binds specifically to CDS.
  • the second antigenbinding domain comprises an scFv.
  • the first antigen-binding domain and the second antigen-binding domain are linked or associated by a covalent bond.
  • the first antigen-binding domain and the second antigen-binding domain are linked by a peptide bond.
  • Certain aspects of the present disclosure are directed to a cell comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a TCR disclosed herein, a recombinant TCR disclosed herein, or a bispecific TCR disclosed herein.
  • the cell further expresses CDS.
  • the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, an natural killer T (NKT) cell, or an ILC cell.
  • the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the an
  • the cancer is relapsed or refractory. In some aspects, the cancer is locally advanced. In some aspects, the cancer is advanced. In some aspects, the cancer is metastatic.
  • the cells are obtained from the subject. In some aspects, the cells are obtained from a donor other than the subject.
  • the subject is preconditioned prior to the administering of the cells.
  • the preconditioning comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof.
  • the preconditioning comprises administering an interleukin.
  • the preconditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof.
  • the preconditioning comprises administering a preconditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, an AKT inhibitor, ATRA, Rapamycin, or any combination thereof.
  • the preconditioning comprises administering cyclophosphamide, fludarabine, or both.
  • Certain aspects of the present disclosure are directed to a method of engineering an antigen-targeting cell, comprising transducing a cell collected from a subject in need of a T cell therapy with a nucleic acid molecule disclosed herein or a vector disclosed herein.
  • the antigen-targeting cell further expresses CD4.
  • the cell is a T cell or a natural killer (NK) cell.
  • Certain aspects of the present disclosure are directed to an HLA class P molecule complexed to a peptide, wherein the HLA class P molecule comprises an alpha chain and a beta chain; and wherein the peptide consists of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the HLA class P molecule is an HLA-DP, HLA-DQ, or HLA-DR allele, or any combination thereof.
  • the HLA class P molecule is an HLA-DP allele.
  • the HLA class P molecule is an HLA-DQ allele.
  • the HLA class P molecule is an HLA-DR allele.
  • the HLA class P molecule is a monomer. In some aspects, the HLA class P molecule is a dimer. In some aspects, the HLA class P molecule is a trimer. In some aspects, the HLA class P molecule is a tetramer. T In some aspects, the HLA class P molecule is a pentamer.
  • Certain aspects of the present disclosure are directed to an antigen presenting cell (APC), comprising an HLA class P molecule disclosed herein.
  • APC antigen presenting cell
  • the HLA class P molecule is expressed on the surface of the APC.
  • Certain aspects of the present disclosure are directed to a method of enriching a target population of T cells obtained from a human subject, comprising contacting the T cells with an HLA class P molecule disclosed herein or an APC disclosed herein, wherein following the contacting, the enriched population of T cells comprises a higher number of T cells capable of binding the HLA class P molecule relative to the number of T cells capable of binding the HLA class P molecule prior to the contacting.
  • Certain aspects of the present disclosure are directed to a method of enriching a target population of T cells obtained from a human subject, comprising contacting the T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as set forth in SEQ ID NO: 13, wherein following the contacting, the enriched population of T cells comprises a higher number of T cells capable of targeting a tumor cell relative to the number of T cells capable of targeting a tumor cell prior to the contacting.
  • the T cells obtained from the human subject are tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • Certain aspects of the present disclosure are directed to a method of enhancing cytotoxic T cell-mediated targeting of cancer cells in a subject afflicted with a cancer, comprising administering to the subject a peptide having an amino acid sequence as set forth in SEQ ID NO: 13.
  • Certain aspects of the present disclosure are directed to a cancer vaccine comprising a peptide having an amino acid sequence as set forth in SEQ ID NO: 13.
  • Certain aspects of the present disclosure are directed to a method of selecting a T cell capable of targeting a tumor cell, comprising contacting a population of isolated T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the T cell is a tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • FIGs. 1A-1B are graphical representations of DP4 L112W/V141M dimer staining of peptide-specific CD4 + T cells from melanoma patients.
  • Primary CD4 + T cells were purified from six DP4 + melanoma patients and stimulated with DP4-expressing aAPCs individually pulsed with CCNDI219-238 peptides and stained with cognate DP4 LU2W/v141M dimers. Examples of DP4 L112W/V141M dimer staining are shown.
  • FIGs. 2A-2D are graphical representations of data illustrating that DP4-restricted
  • FIG. 3 is a bar graph illustrating the results of IL-2 EPISPOT assays of 03-
  • FIGs. 4A-4E are graphical representations of data showing that DP4-restricted CCND1 21 9- 2 38 TCRs isolated from DP4 L112W/V141M dimer-positive cells and reconstituted in human primary CD4 + T cells were functional in a DP4-restricted and antigen-specific manner.
  • 03-CCND1 21 9- 2 38 were retrovirally transduced into human primary CD4 + T cells and stained with the respective DP4 LU2W/V141M dimers. *P ⁇ 0.05 by Student’s t-test. Bars and error bars represent the mean ⁇ SD of results in triplicate experiments (FIG. 4E).
  • FIGs. 5A-5B present data showing that DP4-restricted CCND1219-238 TCRs cloned from melanoma patients recognized peptides endogenously processed and presented by K562-based aAPCs.
  • FIG. 5A is an image of a Western blot analysis showing CCND1 endogenously expressed in K562-derived aAPC cells.
  • FIG. 5B is a bar graph showing the results of IFN-g ELISPOT assays of human primary T cells retrovirally transduced with 03- CCND 1219-238 and stimulated with peptide-unpulsed HLA-null or DP4-aAPCs. *, P ⁇ 0.05 by Student’s t-test. Bars and error bars represent the mean ⁇ SD of results in triplicate experiments.
  • the present disclosure is directed to TCRs or antigen binding portions thereof that specifically bind to an epitope on CCND1, nucleic acid molecules that encode the same, and cells that comprise the TCR or the nucleic acid molecule. Some aspects of the present disclosure are directed to methods of treating a cancer in a subject in need thereof. Other aspects of the present disclosure are directed to HLA class P molecules complexed to a peptide comprising the epitope of CCND1.
  • a or “an” entity refers to one or more of that entity; for example, "a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B, “ “ A” (alone), and “B” (alone).
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non- parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • T cell receptor refers to a heteromeric cell- surface receptor capable of specifically interacting with a target antigen.
  • TCR includes but is not limited to naturally occurring and non-naturally occurring TCRs; full-length TCRs and antigen binding portions thereof; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In human, TCRs are expressed on the surface of T cells, and they are responsible for T cell recognition and targeting of antigen presenting cells.
  • Antigen presenting cells display fragments of foreign proteins (antigens) complexed with the major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class P molecule).
  • MHC major histocompatibility complex
  • a TCR recognizes and binds to the peptide:HLA complex and recruits CDS (for MHC Class I molecules) or CD4 (for MHC class P molecules), activating the TCR.
  • CDS for MHC Class I molecules
  • CD4 for MHC class P molecules
  • a TCR can comprise two chains, an alpha chain and a beta chain (or less commonly a gamma chain and a delta chain), interconnected by disulfide bonds.
  • Each chain comprises a variable domain (alpha chain variable domain and beta chain variable domain) and a constant region (alpha chain constant region and beta chain constant region).
  • the variable domain is located distal to the cell membrane, and the variable domain interacts with an antigen.
  • the constant region is located proximal to the cell membrane.
  • a TCR can further comprises a transmembrane region and a short cytoplasmic tail.
  • the term “constant region” encompasses the transmembrane region and the cytoplasmic tail, when present, as well as the traditional "constant region.”
  • variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each alpha chain variable domain and beta chain variable domain comprises three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDRS, FR4.
  • Each variable domain contains a binding domain that interacts with an antigen. Though all three CDRs on each chain are involved in antigen binding, CDRS is believed to be the primary antigen binding region, while CDR1 and CDR2 are believed to primarily recognize the HLA molecule.
  • TCR also includes an antigen-binding fragment or an antigen-binding portion of any TCR disclosed herein, and includes a monovalent and a divalent fragment or portion, and a single chain TCR.
  • TCR is not limited to naturally occurring TCRs bound to the surface of a T cell.
  • TCR further refers to a TCR described herein that is expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or that is modified to express CD4, as described herein), or a TCR described herein that is free from a cell membrane (e.g., an isolated TCR or a soluble TCR).
  • An "antigen binding molecule,” “portion of a TCR,” or “TCR fragment” refers to any portion of an TCR less than the whole.
  • An antigen binding molecule can include the antigenic CDRs.
  • an "antigen” refers to any molecule, e.g., a peptide, that provokes an immune response or is capable of being bound by a TCR.
  • An “epitope,” as used herein, refers to a portion of a polypeptide that provokes an immune response or is capable of being bound by a TCR.
  • the immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • An antigen and/or an epitope can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed.
  • an antigen and/or an epitope can be specific to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one aspect, antigens are tumor antigens.
  • An epitope can be present in a longer polypeptide (e.g., in a protein), or an epitope can be present as a fragment of a longer polypeptide.
  • an epitope is complexed with a major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class 1 molecule).
  • MHC major histocompatibility complex
  • CCND1 Gl/S-specific cyclin-Dl
  • B-cell lymphoma 1 protein BCL-1
  • PRAD1 refers to a human regulatory component of the cyclin D1-CDK4 (DC) complex that phosphorylates and inhibits members of the retinoblastoma (RB) protein family including RBI and regulates the cell-cycle during Gl/S transition. Phosphorylation of RBI allows dissociation of the transcription factor E2F from the RB/E2F complex and the subsequent transcription of E2F target genes which are responsible for the progression through the G1 phase. CCND1 is also involved in hypophosphorylation of RBI in early G1 phase.
  • Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals.
  • CCND1 is also a substrate for SMAD3, phosphorylating SMAD3 in a cell-cycle-dependent manner and repressing its transcriptional activity.
  • CCND1 is also a component of the ternary complex, cyclin D1/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex, and CCND1 exhibits transcriptional corepressor activity with INSM1 on the NEUROD1 and INS promoters in a cell cycle-independent manner. Mutations, amplification, and overexpression of CCND1, which alter cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis.
  • CCND1 refers to not only the full-length canonical sequence, but also variants and fragments thereof.
  • the amino acid sequence of CCND1 (SEQ ID NO: 16) is provided in Table 1 (UniProtKB - P24385).
  • HLA refers to the human leukocyte antigen.
  • HLA genes encode the major histocompatibility complex (MHC) proteins in humans. MHC proteins are expressed on the surface of cells, and are involved in activation of the immune response.
  • HLA class P genes encode MHC class P proteins which are expressed on the surface of professional antigen presenting cells (APCs).
  • APCs professional antigen presenting cells
  • professional APCs include monocytes, macrophages, dendritic cells (DCs), and B lymphocytes.
  • Some endothelial and epithelial cells can also express MHC class P molecules after inflammatory signals are activated. Humans lacking functional MHC class P molecules are extremely susceptible to an array of infectious diseases and typically die at a young age.
  • an "HLA class P molecule” or “MHC class P molecule” refers to a protein product of a wild-type or variant HLA class P gene encoding an MHC class P molecule. Accordingly, "HLA class P molecule” and “MHC class P molecule” are used interchangeably herein.
  • a typical MHC Class P molecule comprises two protein chains: an alpha chain and a beta chain. In general, naturally occurring alpha chains and beta chains each comprise a transmembrane domain, which anchors the alpha/beta chain to the cell surface, and an extracellular domain, which carries the antigen and interacts with a TCR and/or CD4 expressed on a T cell.
  • Both the MHC Class P alpha and beta chains are encoded by the HLA gene complex.
  • the HLA complex is located within the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes of diverse function.
  • the HLA gene complex is highly variant, with over 20,000 HLA alleles and related alleles, including over 250 MHC class P alpha chain alleles and 5,000 MHC class P beta chain alleles, known in the art, encoding thousands of MHC class P proteins (see, e.g., hla.alleles.org, last visited May 20, 2019, which is incorporated by reference herein in its entirety).
  • DP4 is the most frequently found allele in many ethnic groups.
  • HLA-DP HLA-DP
  • HLA- DQ HLA-DR
  • HLA-DO and HLA-DM encode proteins that associate with the MHC class P molecule and support its configuration and function.
  • the MHC class P molecule When the MHC class P molecule is complexed with an antigen peptide, the 10-30 amino acid long antigen peptide binds the peptide-binding groove and is presented extracellulariy to CD4+ cells. Both the alpha- and beta-chains fold into two separate domains; alpha-1 and alpha-2 for the alpha polypeptide, and beta-1 and beta-2 for the beta polypeptide. The open-ended peptide-binding groove which holds the presented antigen is found between the alpha-1 and beta-1 domains.
  • TCR T cell receptor
  • the beta chain of the MHC class P molecule weakly interacts (K D > 2 mM) with CD4 expressed on the surface of the T cell.
  • the canonical CD4 amino acid sequence (UniProt - P01730) is provided in Table 2 (SEQ ID NO: 17).
  • autologous refers to any material derived from the same individual to which it is later to be re-introduced.
  • an autologous T cell therapy comprises administering to a subject a T cell that was isolated from the same subject.
  • allogeneic refers to any material derived from one individual which is then introduced to another individual of the same species.
  • an allogeneic T cell transplantation comprises administering to a subject a T cell that was obtained from a donor other than the subject.
  • a “cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • a “cancer” or “cancer tissue” can include a tumor. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system including lymphoma, leukemia, and other leukocyte malignancies.
  • the methods of the present invention can be used to reduce the tumor size of a tumor derived from, for example, bone cancer, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non- Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the e
  • NHL
  • a refractory cancer refers to a cancer that is not amendable to surgical intervention, and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.
  • an "anti-tumor effect” as used herein refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor.
  • An anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.
  • progression-free survival which can be abbreviated as PFS, as used herein refers to the time from the treatment date to the date of disease progression per the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.
  • Disease progression or “progressive disease,” which can be abbreviated as PD, as used herein, refers to a worsening of one or more symptom associated with a particular disease.
  • disease progression for a subject afflicted with a cancer can include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.
  • a "cytokine,” as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell.
  • a cytokine can be endogenously expressed by a cell or administered to a subject. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins.
  • homeostatic cytokines including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response.
  • homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma.
  • pro-inflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • sICAM-1 soluble intercellular adhesion molecule 1
  • sVCAM-1 soluble vascular adhesion molecule 1
  • VEGF vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • PLGF placental growth factor
  • effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin.
  • acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).
  • chemokines are a type of cytokine that mediates cell chemotaxis, or directional movement.
  • chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1a (MIP-la, MIP-la), MIP- 1b (MIP-lb), gamma-induced protein 10 (IP-10), and thymus and activation regulated chemokine (TARC or CCL17).
  • analytes and cytokines of the present invention include, but are not limited to chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-
  • G- CSF granulocyte colony-stimulating factor
  • LIF leukemia inhibitory factor
  • OSM oncostatin M
  • CD 154 lymphotoxin (LT) beta
  • 4-1BB ligand (4-1BBL) a proliferation-inducing ligand (APRIL)
  • CD70, CD153, CD178 glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF -related apoptosis-inducing ligand (TRAIL).
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • NK cells include natural killer (NK) cells, T cells, or B cells.
  • NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses. It works through the process of apoptosis or programmed cell death. They were termed“natural killers” because they do not require activation in order to kill cells.
  • T-cells play a major role in cell-mediated-immunity (no antibody involvement).
  • T-cell receptors (TCR) differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the T cell’s maturation.
  • T-cells There are six types of T-cells, namely: Helper T-cells (e.g CD4+ cells), Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killer T cell), Memory T-cells ((i) stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2RP, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytok
  • B-cells play a principal role in humoral immunity (with antibody involvement).
  • a B cell makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction.
  • APCs antigen-presenting cells
  • immature B-cells are formed in the bone marrow, where its name is derived from.
  • the term "genetically engineered” or “engineered” refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof.
  • the cell that is modified is a lymphocyte, e.g., a T cell or a modified cell that expresses CD4, which can either be obtained from a patient or a donor.
  • the cell can be modified to express an exogenous construct, such as, e.g., a T cell receptor (TCR) disclosed herein, which is incorporated into the cell's genome.
  • TCR T cell receptor
  • the cell is modified to express CD4.
  • an "immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • a cell of the immune system for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils
  • soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results
  • immunotherapy refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
  • immunotherapy include, but are not limited to, T cell therapies.
  • T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.
  • T cells used in an immunotherapy described herein can come from any source known in the art.
  • T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject.
  • T cells can be obtained from, e.g., peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • the T cells can be derived from one or more T cell lines available in the art.
  • T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation and/or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No. 2013/0287748, which is herein incorporated by references in its entirety.
  • An immunotherapy can also comprise administering a modified cell to a subject, wherein the modified cell expresses CD4 and a TCR disclosed herein. In some aspects, the modified cell is not a T cell.
  • a "patient” as used herein includes any human who is afflicted with a cancer (e.g., a lymphoma or a leukemia).
  • a cancer e.g., a lymphoma or a leukemia.
  • subject and patient are used interchangeably herein.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule with its cognate ligand, wherein the binding mediates a signal transduction event.
  • a "stimulatory molecule” is a molecule on a T cell, e.g., the T cell receptor (TCR)/CD4 complex, that specifically binds with a cognate stimulatory ligand present on an antigen present cell.
  • a "stimulatory ligand” is a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
  • Stimulatory ligands include, but are not limited to, an MHC Class P molecule loaded with a peptide, an anti-CD4 antibody, a superagonist anti-CD2 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD3 antibody.
  • conditioning and “pre-conditioning” are used interchangeably herein and indicate preparing a patient in need of a T cell therapy for a suitable condition.
  • Conditioning includes, but is not limited to, reducing the number of endogenous lymphocytes, removing a cytokine sink, increasing a serum level of one or more homeostatic cytokines or pro-inflammatory factors, enhancing an effector function of T cells administered after the conditioning, enhancing antigen presenting cell activation and/or availability, or any combination thereof prior to a T cell therapy.
  • conditioning comprises increasing a serum level of one or more cytokines, e.g., interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof.
  • cytokines e.g., interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-induced protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP-1), placental growth factor (PLGF), C-reactive protein (CRP), soluble intercellular
  • Treatment or “treating” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
  • treatment or “treating” includes a partial remission.
  • treatment or “treating” includes a complete remission.
  • the terms "about” or “comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about” or “comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art.
  • “about” or “comprising essentially of' can mean a range of up to 10% (i.e., ⁇ 10%).
  • about 3mg can include any number between 2.7 mg and 3.3 mg (for 10%).
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • the meaning of "about” or “comprising essentially of' should be assumed to be within an acceptable error range for that particular value or composition.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.
  • the present disclosure is directed to T Cell Receptors (TCRs) or antigen binding portions thereof that specifically bind to an epitope on CCND1, nucleic acid molecules that encode the same, and cells that comprise the TCR or the nucleic acid molecule.
  • TCRs T Cell Receptors
  • Some aspects of the present disclosure are directed to methods of treating a cancer in a subject in need thereof, comprising administering to the subject a cell comprising the TCRs described herein.
  • Other aspects of the present disclosure are directed to an epitope of CCND1 that the TCRs bind to and HLA class P molecules complexed to a peptide comprising the epitope of CCND1.
  • TCR The T-cell receptor, or TCR, is a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the binding between TCR and antigen peptides is of relatively low affinity and is degenerate: that is, many TCRs recognize the same antigen peptide and many antigen peptides are recognized by the same TCR.
  • the TCR is composed of two different protein chains (that is, it is a heterodimer).
  • the TCR In 95% of human T cells, the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of human T cells, the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively). This ratio changes during ontogeny and in diseased states (such as leukemia). It also differs between species. Orthologues of the 4 loci have been mapped in various species. Each locus can produce a variety of polypeptides with constant and variable regions.
  • the T lymphocyte When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.
  • signal transduction that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.
  • nucleic acid molecules comprising (i) a first nucleotide sequence encoding a recombinant TCR or an antigen binding portion thereof that specifically binds human CCND1 ("anti-CCNDl TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR.
  • the second nucleotide sequence is a non-naturally occurring sequence. In other aspects, the second nucleotide sequence is synthetic.
  • the second nucleotide sequence comprises a sequence that targets a nucleotide sequence encoding the endogenous TCR.
  • the anti-CCNDl TCR cross competes for binding to human CCND1 with a reference TCR. In some aspects, the anti-CCNDl TCR binds the same epitope or an overlapping epitope of human CCND1 as a reference TCR.
  • the reference TCR comprises an alpha chain and a beta chain; wherein the alpha chain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3; wherein the beta chain comprises a CDR1, a CDR2, and a CDR3; and wherein the reference TCR comprises the alpha chain CDR3 set forth in SEQ ID NO: 7 and the beta chain CDR3 set forth in SEQ ID NO: 10.
  • reference TCR comprises the beta chain CDR1, CDR2, and CDR3 sequences present in the amino acid sequence set forth in SEQ ID NO: 2.
  • the reference TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • the present disclosure is directed to a TCR encoded by the first nucleotide sequence described herein.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain comprises variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3.
  • the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7 (CAVCTLYNFNKFYF).
  • the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10 (CASLTDNNEQFF).
  • the non-CDR regions in the alpha chain and/or the beta chain are further modified, e.g., substitution or mutation of one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, or six amino acids, thereby the alpha chain and/or the beta chain are not naturally occurring.
  • the substitutions or mutations can improve the TCRs described herein in various ways, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 5 (VSNAYN).
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 8 (MNHEY).
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 6 (GSKP).
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 9 (SVGAGI).
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti- CCND1 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises an alpha constant region that is different from endogenous, e.g., naturally occurring, constant regions of the alpha chain.
  • the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises a beta constant region that is different from endogenous, e.g., naturally occurring, constant regions of the beta chain.
  • the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7.
  • the anti- CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti- CCNDl TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within the target sequence relative to the corresponding amino acid sequence of an endogenous TCR.
  • the alpha chain of the anti-CCNDl TCR encoded by the first nucleotide sequence further comprises a signal peptide.
  • Any signal peptide can be used in the anti-CCDNl TCR alpha chains disclosed herein.
  • the signal peptide is a naturally occurring TCR alpha chain signal peptide.
  • the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 21.
  • the signal peptide is a heterologous signal peptide, e.g., a signal peptide derived from a protein other than a TCR alpha chain.
  • the signal peptide is a synthetic signal peptide.
  • the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 20 or 22.
  • the alpha chain of CCND1 TCR encoded by the first nucleotide sequence does not comprise a signal peptide.
  • the signal peptide of the alpha chain is encoded by a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 23 or 24.
  • the beta chain of the anti-CCNDl TCR encoded by the first nucleotide sequence further comprises a signal peptide.
  • Any signal peptide can be used in the anti-CCDNl TCR beta chains disclosed herein.
  • the signal peptide is a naturally occurring TCR beta chain signal peptide.
  • the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 22.
  • the signal peptide is a heterologous signal peptide, e.g., a signal peptide derived from a protein other than a TCR beta chain.
  • the signal peptide is a synthetic signal peptide.
  • the signal peptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 20 or 21.
  • the beta chain of CCND1 TCR encoded by the first nucleotide sequence does not comprise a signal peptide.
  • the signal peptide of the beta chain is encoded by a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 23 or 24.
  • each of the alpha chain and the beta chain of the anti-CCNDl TCR encoded by the first nucleotide sequence further comprises a signal peptide.
  • the signal peptide of the alpha chain is the same as the signal peptide of the beta chain.
  • the signal peptide of the alpha chain is different from the signal peptide of the beta chain.
  • the anti-CCNDl TCR encoded by the first nucleotide sequence binds the same epitope as a reference TCR.
  • the anti-CCNDl TCR binds to an epitope of CCNDl comprising the amino acid sequence set forth in SEQ ID NO: 13 (SPNNFLSYYRLTRFLSRVIK).
  • the anti-CCNDl TCR binds to an epitope of CCNDl consisting of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the epitope consists of amino acid residues 219-238 of CCNDl (SEQ ID NO: 16), e.g., "CCNDl 219-238 .”
  • the epitope is part of a larger polypeptide, e.g., a peptide that comprises the epitope sequence and (i) one or more additional amino acids N-terminal to the epitope sequence and/or (ii) one or more additional amino acids C-terminal to the epitope sequence.
  • the polypeptide comprising the epitope is at least about 10 amino acids, at least about 11 amino acids, at least about 12 amino acids, at least about 13 amino acids, at least about 14 amino acids, at least about 15 amino acids, at least about 16 amino acids, at least about 17 amino acids, at least about 18 amino acids, at least about 19 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 45 amino acids, or at least about 50 amino acids in length.
  • the polypeptide comprising the epitope is at least about 5 to at least about 10, at least about 5 to at least about 15, at least about 5 to at least about 20, at least about 10 to at least about 15, at least about 10 to at least about 20, at least about 10 to at least about 25, at least about 10 to at least about 30, at least about 10 to at least about 35, at least about 10 to at least about 40, at least about 10 to at least about 45, at least about 10 to at least about 50, at least about 15 to at least about 20, at least about 15 to at least about 25, at least about 15 to at least about 30, at least about 15 to at least about 35, at least about 15 to at least about 40, at least about 15 to at least about 45, or at least about 15 to at least about 50 amino acids in length.
  • the polypeptide comprising the epitope comprises the epitope and at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 additional amino acids N-terminal to the epitope.
  • the polypeptide comprising the epitope comprises the epitope and at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15 additional amino acids C-terminal to the epitope.
  • the epitope is complexed with an HLA class P molecule.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • TCR T-cell receptor
  • Class P MHC molecules are present as transmembrane glycoproteins on the surface of professional antigen presenting cells (APCs).
  • Intact class P molecules consist of an alpha chain and a beta chain.
  • the gene encoding the alpha chain of MHC P class molecules is composed of 5 exons, and the gene encoding the beta chain is composed of 6 exons.
  • Exon 1 encodes the leader peptide
  • exons 2 and 3 encode the two extracellular domains
  • exons 4 and 5 contribute to the transmembrane domain and cytoplasmic tail for each of the alpha and beta subunits.
  • Three loci in the HLA complex encode MHC class P proteins: HLA-DR, HLA-DQ, and HLA-DP.
  • CD4 T cells that express CD4 molecules react with class P MHC molecules. These lymphocytes often have a cytotoxic ftmction and activate a response to eliminate self-cells infected with intracellular pathogens or to destroy extracellular parasites. Because only professional antigen presenting cells (APCs) express class P MHC molecules, only these cells present antigen for CD4 T cells (CD4 binds to the nonpolymorphic part of the alpha-2 and beta-2 domains of the alpha and beta chains of an MHC class P molecule respectively).
  • APCs professional antigen presenting cells
  • the HLA class P alpha and beta chains are selected from an HLA-DR, HLA-DP, and HLA-DQ allele.
  • the HLA class P alpha chain is an HLA-DR alpha chain.
  • the HLA class P beta chain is an HLA-DR beta chain.
  • the HLA class P alpha chain is an HLA-DP alpha chain.
  • the HLA class P beta chain is an HLA-DP beta chain.
  • the HLA class P alpha chain is an HLA-DQ alpha chain.
  • the HLA class P beta chain is an HLA-DQ beta chain.
  • HLA-DR HLA-DR
  • HLA-DP HLA-DP
  • HLA-DQ HLA-DQ alleles
  • the second nucleotide sequence of the nucleic acid molecule disclosed herein can be any sequence or can encode for any polypeptide that is capable of inhibiting the expression of an endogenous TCR.
  • the second nucleotide sequence is one or more siRNAs.
  • the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of an endogenous TCR.
  • the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of wild-type, human TCR.
  • the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the alpha chain of wild-type TCR. In some aspects, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the beta chain of wild-type TCR.
  • the one or more siRNAs comprise (i) one or more siRNA's that are complementary to a target sequence within a nucleotide sequence encoding a constant region of the alpha chain of wild- type TCR and (ii) one or more siRNA's that are complementary to a target sequence within a nucleotide sequence encoding a constant region of the beta chain of wild-type TCR.
  • the one or more siRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 25-28 (Table 4).
  • the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the alpha chain of wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 25 and 26.
  • the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the beta chain of wild- type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 27 and 28.
  • the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs comprise (i) one or more siRNAs that are complementary to a target sequence within a nucleotide sequence encoding a constant region of the alpha chain of wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 25 and 26; and (ii) one or more siRNAs that are complementary to a target sequence within a nucleotide sequence encoding a constant region of the beta chain of wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 27 and 28.
  • the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NOs: 25-28. In some aspects, the second nucleotide sequence comprises SEQ ID NOs: 25-28, wherein one or more of SEQ ID NOs: 25-28 is separated by one or more nucleic acids that do not encode an siRNA. In certain aspects, the one or more siRNAs are selected from the siRNAs disclosed in U.S. Publication No. 2010/0273213 Al, which is incorporated by reference herein in its entirety.
  • the second nucleotide sequence of the nucleic acid molecule encodes a protein, wherein the protein is capable of inhibiting the expression of an endogenous, e.g., wild-type, TCR.
  • the second nucleotide sequence encodes Cas9.
  • the vector is a viral vector.
  • the vector is a viral particle or a virus.
  • the vector is a mammalian vector.
  • the vector is a bacterial vector.
  • the vector is a retroviral vector.
  • the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, and an adeno associated virus (AAV) vector.
  • the vector is an AAV vector.
  • the vector is a lentivirus.
  • the vector is an AAV vector.
  • the vector is a Sendai virus.
  • the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present disclosure can be found in Huang and Kamihira, Biotechnol. Adv. 5/(2):208-23 (2103), which is incorporated by reference herein in its entirety.
  • TCRs T Cell Receptors
  • Certain aspects of the present disclosure are directed to recombinant T cell receptors (TCRs) or an antigen binding portion thereof that specifically bind human CCND1 ("an anti-CCNDl TCR").
  • TCRs T cell receptors
  • an antigen binding portion thereof that specifically bind human CCND1 (“an anti-CCNDl TCR”).
  • the anti-CCNDl TCR is encoded by the a nucleic acid molecule disclosed herein.
  • the anti-CCNDl TCR cross competes for binding to human CCND1 with a reference TCR.
  • the anti-CCNDl TCR binds the same epitope or an overlapping epitope of human CCND1 as a reference TCR.
  • the reference TCR comprises an alpha chain and a beta chain, and the alpha chain comprises of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 1.
  • the beta chain of the reference TCR comprises an amino acid sequence as set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein the alpha chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein the beta chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region, and wherein the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1; and (ii) the beta chain constant region comprises an amino acid sequence having a least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the alpha chain of the anti-CCNDl TCR comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and the beta chain of the anti-CCNDl TCR comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3.
  • the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7.
  • the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the alpha chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 5.
  • the beta chain CDR1 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 8.
  • the alpha chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 6.
  • the beta chain CDR2 of the anti-CCNDl TCR comprises an amino acid sequence as set forth in SEQ ID NO: 9.
  • the anti-CCNDl TCR comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7. In some aspects, the anti-CCNDl TCR comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti-CCNDl TCR comprises a beta chain variable domain present in the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region.
  • the anti-CCNDl TCR comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7. In some aspects, the anti-CCNDl TCR comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises an alpha constant region that is different from endogenous, e.g., naturally occurring, constant regions of the alpha chain.
  • the alpha chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with a constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, or at least about 99% sequence identity with a constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the anti-CCNDl TCR comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR encoded by the first nucleotide further comprises a beta constant region that is different from endogenous, e.g., naturally occurring, constant regions of the beta chain.
  • the beta chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the alpha chain amino acid sequence set forth in SEQ ID NO: 1.
  • the anti- CCNDl TCR comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the alpha chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-CCNDl TCR comprises an alpha chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 7. In some aspects, the anti- CCNDl TCR comprises an alpha chain comprising the amino add sequence set forth in SEQ ID NO: 1.
  • the anti-CCNDl TCR comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the beta chain amino acid sequence set forth in SEQ ID NO: 2.
  • the anti- CCNDl TCR comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, or about 100% sequence identity with the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-CCNDl TCR comprises a beta chain CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 10. In some aspects, the anti- CCNDl TCR comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the anti-CCNDl TCR comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within the target sequence relative to the corresponding amino acid sequence of an endogenous TCR.
  • the anti-CCNDl TCR binds the same epitope as a reference TCR. In some aspects, the anti-CCNDl TCR binds to an epitope of CCND1 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the anti-CCNDl TCR binds to an epitope of CCND1 consisting of an amino acid sequence as set forth in SEQ ID NO: 13. In some aspects, the epitope consists of amino acid residues 219-238 of CCND1 (SEQ ID NO: 16), e.g., " CCND1 219-238 .”
  • the epitope is complexed with an HLA class P molecule.
  • the HLA class P molecule comprises an alpha chain and a beta chain.
  • the alpha chain is selected from an HLA-DR alpha chain, an HLA-DP alpha chain, and an HLA-DQ alpha chain.
  • the beta chain is selected from an HLA-DR beta chain, an HLA-DP beta chain, and an HLA-DQ beta chain.
  • the HLA class P molecule comprises an HLA-DR alpha chain and an HLA-DR beta chain.
  • the HLA class P molecule comprises an HLA-DP alpha chain and an HLA-DP beta chain.
  • the HLA class P molecule comprises an HLA-DQ alpha chain and an HLA-DQ beta chain.
  • HLA-DR HLA-DR
  • HLA-DP HLA-DP
  • HLA-DQ HLA-DQ alleles
  • TCRs Bispecific T Cell Receptors
  • Certain aspects of the present disclosure are directed to a bispecific TCR comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a TCR or an antigen-binding portion thereof disclosed herein.
  • the first antigen-binding domain comprises a single chain variable fragment ("scFv").
  • the second antigen-binding domain binds specifically to a protein expressed on the surface of a T cell. Any protein expressed on the surface of a T cell can be targeted by the bispecific antibody disclosed herein. In certain aspects, the protein expressed on the surface of a T cell is not expressed by other cells. In some aspects, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells. In some aspects, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells, but it is not expressed on the surface of a human non-immune cell.
  • the second antigen-binding domain binds specifically to a protein expressed on the surface of a T cell selected from CD3, CD4, CD2, CDS, CD6, CDS, CDlla (LFA-1a), CD43, CD45, and CD53. In certain aspects, the second antigen-binding domain binds specifically to CD3. In certain aspects, the second antigen-binding domain binds specifically to CD4. In some aspects, the second antigenbinding domain comprises an scFv.
  • first antigen-binding domain and the second antigen-binding domain are linked or associated by a covalent bond. In some aspects, the first antigen-binding domain and the second antigen-binding domain are linked by a peptide bond.
  • Certain aspects of the present disclosure are directed to cells comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, or any combination thereof. Any cell can be used in the present disclosure.
  • the cell expresses CD4.
  • CD4 expression can be naturally occurring, e.g., the CD4 is expressed from a nucleic acid sequence that is endogenously expressed by the cell.
  • T cells, monocytes, macrophages, dendritic cells, and natural killer (NK) cells naturally express CD4.
  • the cell is a T cell, a monocyte, a macrophage, a dendritic cell, or a natural killer cell.
  • the cell is a T cell selected from a natural killer T (NKT) cell and an innate lymphoid cell (ILC).
  • the cell is a monocyte.
  • the cell is a macrophage.
  • the cell is a dendritic cell.
  • the T cell is isolated from a human subject.
  • the human subject is the same subject that will ultimately receive the T cell therapy.
  • the subject is a donor subject, wherein the donor subject is not the same subject that will receive the T cell therapy.
  • the cell is a cell that does not naturally express CD4, wherein the cell has been modified to express CD4.
  • the cell comprises a transgene encoding CD4, wherein the transgene is expressed by the cell.
  • the cell comprises a transgene encoding a protein that activates expression of endogenous CD4 by the cell.
  • the cell comprises a transgene encoding a protein or siRNA that inhibits an inhibitor of CD4 expression in the cell.
  • the transgene is incorporated into the genome of the cell. In some aspects, the transgene is not incorporated into the genome of the cell.
  • the cell that is modified to express CD4 is isolated from a human subject.
  • the human subject is the same subject that will ultimately receive the cell therapy.
  • the subject is a donor subject, wherein the donor subject is not the same subject that will receive the cell therapy.
  • Certain aspects of the present disclosure are directed to a HLA class P molecule complexed to a peptide, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
  • the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13.
  • the HLA Class P molecule is an HLA-DR, HLA-DP, or an HLA- DQ allele. In some aspects, the HLA class P molecule is any HLA allele disclosed at hla.alleles.org/ (last visited on February 27, 2019)
  • the HLA Class P molecule comprises an alpha chain and a beta chain.
  • the sequence of the alpha chain is selected from any of the HLA alpha chain protein sequences available at hla.alleles.org (last visited February 27, 2019).
  • the alpha chain is an HLA-DP alpha chain. Any HLA-DP alpha chain allele known in the art can be used in the compositions and methods disclosed herein.
  • the alpha chain is selected from an HLA-DPA1*01, HLA-DPA1*02, HLA- DP A1 *03, and HLA-DPA1*04 allele.
  • the DP alpha chain comprises an HLA-DPA1*01 allele.
  • the DP alpha chain comprises an HLA-DPA1*02 allele.
  • the DP alpha chain comprises an HLA-DPA1*03 allele.
  • the DP alpha chain comprises an HLA-DP A1 *04 allele.
  • the DP alpha chain is selected from DPA1 *01:03:01:01, DPA1 *01:03:01:02, DPA1 *01:03:01:03, DPA1 *01:03:01:04, DPA1 *01:03:01:05,
  • DPA1 *01:03:01: 14 DPA1*01:03:01:15, DPA1*01:03:01:16, DPA1 *01:03:01: 17,
  • DPA1*01:03:01:18Q DPA1 *01:03:01: 19, DPA1 *01:03:01:20, DPA1*01:03:01:21, DPA1 *01:03:01:22, DPA1 *01:03:01:23, DPA1 *01:03:02, DPA1 *01:03:03, DPA1 *01:03:04, DPA1 *01:03:05, DPA1*01:03:06, DPA1 *01 :03:07, DPA1 *01 :03:08, DPA1 *01 :03:09, DPA1 *01:04, DPA1 *01:05, DPA1 *01:06:01, DPA1 *01:06:02, DPA1 *01:07, DPA1 *01:08, DPA1 *01:09, DPA1*01:10, DPA1 *01:11, DPA1 *01:12, DPA1*01:13, DPA1*01:14,
  • DPA1 *02:01:01: 10 DPA1 *02:01:01: 11, DPA1 *02:01:02:01, DPA1 *02:01:02:02,
  • the beta chain is an HLA-DP beta chain. Any HLA-DP beta chain allele known in the art can be used in the compositions and methods disclosed herein.
  • the DP beta chain comprises an allele selected from an DPB1*01, DPB1*02, DPB1*03, DPB1 *04, DPB1*05, DPB1*06, DPB1*08, DPB1*09, DPB1*10, DPB1*100, DPB1*101, DPB1*102, DPB1*103, DPB1*104, DPB1*105, DPB1*106,
  • DPB1*235 DPB 1*236, DPB1*237, DPB1*238, DPB1*239, DPB1*24, DPB1*240,
  • DPB 1*400 DPB1*401, DPB1*402, DPB1*403, DPB1*404, DPB1*405, DPB1*406, DPB 1*407, DPB 1*408, DPB1*409, DPB1*41, DPB1*410, DPB1*411, DPB1*412,
  • DPB 1*638 DPB 1*639, DPB 1*64, DPB 1*640, DPB1*641, DPB 1*642, DPB 1*643,
  • DPB1*810 DPB 1*811, DPB 1*812, DPB 1*813, DPB 1*814, DPB 1*815, DPB1*816,
  • the DP beta chain comprises an HLA-DPB1*01, HLA-DPB1*02, HLA-DPB1*01, HLA- DPB1*03, HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*06, HLA-DPB1*08, HLA- DPB1*09 allele, and any combination thereof.
  • the DP beta chain comprises an HLA-DPB1*04 allele.
  • the DP beta chain comprises an HLA-DPB1*04:01 allele.
  • the DP beta chain comprises an allele selected from DPB1*01:01:01:01, DPB1 *01:01:01:02, DPB1*01:01:01:03, DPB1*01:01:01:04,
  • DPB1*02:01:02:06 DPB1*02:01:02:07, DPB1*02:01:02:08, DPB1*02:01:02:09,
  • DPB 1*02:01:02: 10 DPB1*02:01:02:11, DPB1 *02:01:02: 12, DPB1*02:01:02:13,
  • DPB1*02:01:02:14 DPB1*02:01:02:15, DPB1*02:01:02:16, DPB1*02:01:02:17,
  • DPB 1*02:01:02:26 DPB 1*02:01:02:27, DPB1*02:01:02:28, DPB1*02:01:02:29,
  • DPB 1*02:01:02:34 DPB 1*02:01:02:35, DPB1*02:01:02:36, DPB1*02:01:02:37,
  • DPB 1*02:02:01:07 DPB 1*02:02:02, DPB 1*02:02:03, DPB1*03:01:01:01,
  • DPB1*03:01:01:06 DPB 1*03:01:07, DPB 1*03:01:08, DPB1*03:01:01:09,
  • DPB1 *04:01:01: 19 DPB1*04:01:01:20, DPB1 *04:01:01:21, DPB1*04:01:01:22,
  • DPB1*04:02:01:01 DPB1*04:02:01:02, DPB1*04:02:01:03, DPB1*04:02:01:04,
  • DPB1*04:02:01:09 DPB1*04:02:01:10, DPB1*04:02:01:11, DPB1*04:02:01:12,
  • DPB 1*104:01:02 DPB1*105:01:01:01, DPB1*105:01:01:02, DPB1*105:01:01:03, DPB1*105:01:01:04, DPB1*105:01:01:05, DPB1*105:01:01:06, DPB1*105:01:01:07,
  • DPB1*15:01:01:02 DPB1*15:01:01:03, DPB1*15:01:01:04, DPB1*15:01:02,
  • DPB1*664:01 DPB1*665:01, DPB1*666:01, DPB1*667:01, DPB1*668:01:01:01,
  • DPB1*936:01Q DPB1*937:01, DPB1*938:01, DPB1*939:01N, DPB1*93:01,
  • the alpha chain is an HLA-DQ alpha chain. Any HLA-DQ alpha chain allele known in the art can be used in the compositions and methods disclosed herein. In some aspects, the alpha chain is selected from an HLA-DQA1*01, HLA-DQA1*02, HLA- DQ A1 *03, HLA-DQA1*04, HLA-DQA1*05, and HLA-DQA1*06 allele.
  • the alpha chain is an HLA-DQA1 allele selected from *01:01:01:01, *01:01:01:02, *01:01:01:03, *01:01:01:05, *01:01:01:06, *01:01:02, *01:01:03, *01:01:04, *01:01:05, *01:02:01:01, *01:02:01:02, *01:02:01:03, *01:02:01:04, *01:02:01:05, *01:02:01:06, *01:02:01:05, *01:02:01:06,
  • the beta chain is an HLA-DQ beta chain. Any HLA-DQ beta chain allele known in the art can be used in the compositions and methods disclosed herein. In some aspects the beta chain is selected from an HLA-DQB1*02, HLA-DQB1*03, HLA- DQB1*04, HLA-DQB1*05, and HLA-DQB 1*06 allele.
  • the DQ beta chain comprises an allele selected from DQB 1*02:01:01, DQB 1*02:01:02, DQB 1*02:01:03, DQB 1*02:01:04, DQB1*02:01:05, DQB1 *02:01:06, DQB1 *02:01:07, DQB1*02:01:08, DQB1*02:01 :09, DQB1*02:01 :10,
  • DQB1*02:01:31 DQB1*02:02:01:01, DQB1*02:02:01:02, DQB1*02:02:01:03,
  • DQB 1*03:01:01: 12 DQB1*03:01:01:14, DQB1*03:01:01:15, DQB1*03:01:01:16,
  • DQB1*03:116 DQB1*03:117, DQB1*03:118N, DQB1*03:119, DQB 1*03: 12,
  • DQB 1*03:225 DQB 1*03:226, DQB 1*03:227, DQB 1*03:228, DQB 1*03:229,
  • DQB 1*03:262 DQB 1*03:263, DQB 1*03:264, DQB 1*03:265, DQB 1*03:266,
  • DQB1*04:01:04 DQB1*04:01:05, DQB1*04:02:01:01, DQB1*04:02:01:04,
  • DQB1 *05:1 ION DQB1*05:111, DQB1*05:112, DQB1*05:113, DQB1*05:114,
  • DQB1*05:115 DQB1*05: 116, DQB1*05: 117, DQB1*05:118, DQB1*05:119,
  • DQB1*05:206N DQB1*05:207, DQBl*05:208N5bp, DQB1*05:209, DQB1*05:21,
  • DQB1*06:102N DQB1*06:103, DQB1*06:104, DQB1*06:105, DQB1*06:106,
  • DQB1*06:112N DQB1*06:113, DQB1*06:114, DQB1*06:115, DQB1*06:116,
  • DQB 1*06:279 DQB1*06:27:01, DQB 1*06:27:02, DQB 1*06:28, DQB 1*06:280, DQB1*06:281, DQB 1*06:282, DQB 1*06:283, DQB 1*06:284, DQB 1*06:285,
  • DQB1*06:306N DQB1*06:307, DQB1*06:308N, DQB1*06:309, DQB1*06:31, DQB1*06:310, DQB1*06:311, DQB1*06:312, DQB1*06:313, DQB1 *06:314,
  • the alpha chain is an HLA-DR alpha chain. Any HLA-DR alpha chain allele known in the art can be used in the compositions and methods disclosed herein. In some aspects, the alpha chain is an HLA-DRA*01 allele. In some aspects, the alpha chain is an HLA-DRA1 allele selected from *01:01:01:01, *01:01:01:02, *01:01:01:03, *01:01:02, *01:02:01, *01:02:02, *01:02:03, and any combination thereof.
  • the beta chain is an HLA-DR beta chain. Any HLA-DR beta chain allele known in the art can be used in the compositions and methods disclosed herein.
  • the beta chain is selected from an HLA-DRB1*01, HLA-DRB1*03, HLA- DRB1*04, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA- DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, and HLA- DRB1*16 allele.
  • the beta chain is a DRB3 allele.
  • the beta chain is a DRB4 allele.
  • the beta chain is a DRB5 allele.
  • the beta chain is selected from DRB1*01:01:01, DRB 1*01:01:02,
  • DRB1*01:01:33 DRB1*01:02:01:01, DRB1*01:02:01:02, DRB1*01:02:02, DRB1*01:02:02,
  • DRB1*01:02:13 DRB1*01:03:01, DRB1*01:03:02, DRB 1*01:03:03, DRB1*01:03:04,
  • DRB1*01:04 DRB1*01:05, DRB1*01:06, DRB1*01:07, DRB1*01:08, DRB1*01:09, DRB1*01:10, DRB1*01:100, DRB1*01:11:01, DRB 1*01:11:02, DRB1*01:12,
  • DRB1*03:01:04 DRB1*03:01:05, DRB1*03:01:06, DRB1*03:01:07, DRB1*03:01:08,
  • DRB1*03:02:01 DRB 1*03:02:02, DRB 1*03:02:03, DRB1*03:03, DRB1*03:04:01,
  • DRB 1*03:04:02 DRB1*03:05:01, DRB 1*03:05:02, DRB 1*03:05:03, DRB1*03:06,
  • DRB1*03:104 DRB1*03:105, DRB1*03:106, DRB1*03:107, DRB1*03:108,
  • DRB1*03:119 DRB1*03:11:01, DRB1*03:12, DRB1*03:120, DRB1*03:121,
  • DRB 1*03: 14 DRB1*03:140, DRB1*03:141, DRB1*03:142, DRB1*03:143, DRB1*03:144, DRB 1*03: 145, DRB 1*03: 146, DRB 1*03: 147, DRB 1*03: 148, DRB 1*03: 149,
  • DRB1*04:01:04 DRB1*04:01:05, DRB1*04:01:06, DRB1*04:01:07, DRB1*04:01:08,
  • DRB1*04:04:01 DRB 1*04:04:02, DRB 1*04:04:03, DRB 1*04:04:04, DRB 1*04:04:05,
  • DRB1 *04:05: 14 DRB 1*04:05: 15, DRB1*04:05:16, DRB1*04:05:17, DRB 1*04:05: 18,
  • DRB1*04:05: 19 DRB 1*04:05:20, DRB1*04:06:01, DRB 1*04:06:02, DRB 1*04:06:03,
  • DRB 1*04:225 DRB 1*04:226:01, DRB 1*04:226:02, DRB1*04:227, DRB1*04:228,
  • DRB 1*07:04 DRB1*07:05, DRB1*07:06, DRB1*07:07, DRB1*07:08, DRB1*07:09,
  • DRB1*07:100 DRB1*07:101N, DRB1*07:10N, DRB1*07:11, DRB1*07:12, DRB1*07:13, DRB1*07:14, DRB1*07:15, DRB1*07:16, DRB1*07:17, DRB1*07: 18, DRB1*07:19,
  • DRB1*07:26N DRB 1*07:27, DRB 1*07:28, DRB 1*07:29, DRB 1*07:30, DRB 1*07:31, DRB 1*07:32, DRB1*07:33, DRB1*07:34, DRB1*07:35, DRB1*07:36, DRB1*07:37,
  • DRB1*07:68N DRB1*07:69, DRB1*07:70, DRB1*07:71, DRB1*07:72, DRB1*07:73, DRB 1*07:74, DRB1*07:75, DRB1*07:76, DRB1*07:77, DRB1*07:78, DRB1*07:79,
  • DRB1*11:04:04 DRB1*11:04:05, DRB1*11:04:06, DRB1*11:04:07, DRB1*11:04:08,
  • DRB1* 11:04: 14 DRB1*11:04: 15, DRB1*11:04: 16, DRB1*11:04: 17, DRB1*11:04:18,
  • DRB1*11:119 DRB1*11:11:01, DRB1*11:11:03, DRB1*11:120, DRB1*11:121,
  • DRB1*11:199 DRB1*11:19:01, DRB1*11:19:02, DRB1*11:19:03, DRB1*11:20,
  • DRB1*11:200 DRB1*11:201, DRB1*11:202, DRB1*11:203, DRB1*11:204,
  • DRB1*11:66:02 DRB1*11:67, DRB1*11:68, DRB1*11:69, DRB1*11:70, DRB1*11:72, DRB1*11:73, DRB1*11:74:01, DRB1*11:74:02, DRB1*11:75, DRB1*11:76, DRB1*11:77, DRB1*11:78, DRB1*11:79, DRB1*11:80, DRB1*11:81, DRB1*11:82, DRB1*11:83,
  • DRB 1*12:02:04 DRB1* 12:02:05, DRB1* 12:02:06, DRB1*12:02:07, DRB1*12:02:08, DRB 1*12:02:09, DRB1* 12:03:02, DRB 1*12:03:03, DRB1*12:04, DRB1*12:05, DRB 1*12:06, DRB1*12:07, DRB1*12:08, DRB1*12:09, DRB1*12:10, DRB1*12:11,
  • DRB1*13:01:04 DRB1*13:01:05, DRB1*13:01:06, DRB1*13:01:07, DRB1*13:01:08,
  • DRB1*13:02:04 DRB1*13:02:05, DRB1*13:02:06, DRB 1*13:02:07, DRB1*13:02:08,
  • DRB1*13:278Q DRB1*13:279, DRB1 *13:28:01, DRB1*13:28:02, DRB1*13:29,
  • DRB1*13:30 DRB1*13:31, DRB1*13:32, DRB1*13:33:01, DRB1*13:33:02,
  • DRB 1*14: 14 DRB1*14:140, DRB1*14:141, DRB1*14:142, DRB1*14:143, DRB1*14:144, DRB1*14:145, DRB1*14:146, DRB1*14:147, DRB1*14:148, DRB1*14: 149, DRB1*14:15, DRB 1*14: 150, DRB1*14:151, DRB1*14:152N, DRB1*14:153, DRB1*14:154,
  • DRB1*15:02:04 DRB1* 15:02:05, DRB1* 15:02:06, DRB1*15:02:07, DRB 1*15:02:08, DRB1* 15:02:09, DRB1*15:02:10, DRB1*15:02:11, DRB1*15:02:12, DRB1*15:02:13,
  • DRB1* 15:02: 14 DRB1*15:02:15, DRB1*15:02:16, DRB1*15:02:17, DRB1*15:02:18,
  • DRB1* 15:02: 19 DRB1*15:03:01:01, DRB1*15:03:01:02, DRB1*15:03:01:03,
  • DRB1*15:112 DRB1*15:113N, DRB1*15:114, DRB1*15:115N, DRB1*15:116,
  • DRB1*15:134N DRB1*15:135, DRB1*15:136, DRB1*15:137N, DRB1*15:138N,
  • DRB1*15:154N DRB1*15:155, DRB1*15:156, DRB1*15:157, DRB1*15:158, DRB1*15:159N, DRB1*15:15:01, DRB1*15:15:02, DRB1*15:15:03, DRB1*15:16, DRB1*15:160, DRB1*15:161, DRB1*15:162, DRB1*15:163N, DRB1*15:164Q,
  • DRB1*15:50N DRB1*15:51, DRB1*15:52, DRB1*15:53, DRB1*15:54, DRB1*15:55, DRB1*15:56, DRB1*15:57, DRB1*15:58, DRB1*15:59, DRB1*15:60, DRB1*15:61,
  • DRB1*16:02:04 DRB1* 16:02:05, DRB1* 16:02:06, DRB1*16:02:07, DRB1* 16:02:08, DRB1*16:03, DRB1*16:04:01, DRB1*16:04:02, DRB1*16:05:01, DRB1*16:05:02,
  • DRB3*01:40:02N DRB3*01:41, DRB3 *01:42, DRB3*01:43, DRB3 *01 :44, DRB3*01:45, DRB3 *01:46, DRB3*01:47, DRB3*01:48, DRB3*01:49, DRB3*01:50, DRB3*01:51,
  • DRB3*03:01:04 DRB3*03:01:05, DRB3*03:01:06, DRB3*03:01:07, DRB3 *03:02,
  • DRB4*01:04 DRB4*01:05, DRB4*01:06, DRB4*01:07:01, DRB4*01:07:02, DRB4*01:08, DRB4*01:09, DRB4*01:10, DRB4*01:11, DRB4*01:12, DRB4*01:13, DRB4*01:14,
  • DRB4*01:93, DRB4*02:01N DRB5*01:01:01, DRB5*01:01:02, DRB5*01:01:02, DRB5*01:01:03, DRB5*01:01:04, DRB5*01:02, DRB5*01:03, DRB5*01:04, DRB5*01:05, DRB5*01:06, DRB5*01:07, DRB5*01:08N, DRB5*01:09, DRB5*01:10N, DRB5*01:11, DRB5*01:12, DRB5*01:13, DRB5*01:14, DRB5*01:15, DRB5*01:16, DRB5*01:17,
  • the HLA class P molecule is a monomer. In some aspects, the HLA class P molecule is a dimer. In some aspects, the HLA class P molecule is a multimer. In some aspects, the HLA class P molecule is a trimer. In some aspects, the HLA class P molecule is a tetramer. In some aspects, the HLA class P molecule is a pentamer.
  • Certain aspects of the present disclosure are directed to antigen presenting cells (APCs) comprising any HLA class P molecule disclosed herein.
  • the APC expressed the HLA class P molecule on the surface of the APC.
  • the APC comprises more than one HLA class P molecule disclosed herein.
  • cancer vaccine comprising a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 13.
  • the cancer vaccine comprises a peptide that consists of the amino add sequence set forth in SEQ ID NO: 13.
  • the vaccine further comprises one or more excipient.
  • the vaccine further comprises one or more additional peptides.
  • the one or more additional peptides comprise one or more additional epitopes.
  • Certain aspects of the present disclosure are directed to methods of treating a cancer in a subject in need thereof. Other aspects of the present disclosure are directed to methods of engineering an antigen-targeting cell. Other aspects of the present disclosure are directed to methods of enriching a target population of T cells obtained from a human subject.
  • Certain aspects of the present disclosure are directed to methods of treating a cancer in a subject in need thereof, comprising administering to the subject a nucleic acid molecule disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, an epitope disclosed herein, or an HLA class P molecule disclosed herein, or a vector or cell comprising any of the above.
  • the cancer is selected from melanoma, bone cancer, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular malignant melanoma, pancreatic cancer, skin cancer, cancer of the head or neck, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of
  • the cancer is relapsed. In some aspects, the cancer is refractory.
  • the cancer is advanced. In some aspects, the cancer is metastatic.
  • the methods disclosed herein treat a cancer in a subject.
  • the methods disclosed herein reduce the severity of one or more symptom of the cancer.
  • the methods disclosed herein reduce the size or number of a tumor derived from the cancer.
  • the methods disclosed herein increase the overall survival of the subject, relative to a subject not provided the methods disclosed herein.
  • the methods disclosed herein increase the progressive-free survival of the subject, relative to a subject not provided the methods disclosed herein.
  • the methods disclosed herein lead to a partial response in the subject.
  • the methods disclosed herein lead to a complete response in the subject.
  • the methods disclosed herein comprise treating a cancer in a subject in need thereof, comprising administering to the subject a cell described herein, wherein the cell comprises a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, and/or a bispecific antibody disclosed herein.
  • the cell is a T cell.
  • the cell is a cell that is modified to express CD4.
  • the cell e.g., a T cell
  • the cell is obtained from the subject.
  • the cell e.g., a T cell
  • the subject is preconditioned prior to administering the cells.
  • the preconditioning can comprise any substance that promotes T cell function and/or survival.
  • the preconditioning comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof.
  • the preconditioning comprises administering an interleukin.
  • the preconditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof.
  • the preconditioning comprises administering cyclophosphamide, fludarabine, or both.
  • the preconditioning comprises administering vitamin C, an AKT inhibitor, ATRA (vesanoid, tretinoin), rapamycin, or any combination thereof.
  • the antigen is a CCND1 antigen.
  • the method comprises transducing a cell with a nucleic acid molecule disclosed herein or a vector disclosed herein.
  • the cell can be any cell described herein.
  • the cell is a T cell described herein.
  • the cell is a cell that is modified to express CD4, as described herein.
  • the cell e.g., the T cell, is obtained from a subject in need of a T cell therapy.
  • the cell is obtained from a donor other than the subject in need of the T cell therapy.
  • the cell is a T cell or a natural killer cell.
  • Certain aspects of the present disclosure are directed to methods of enriching a target population of T cells obtained from a human subject.
  • the method comprises contacting the T cells with an HLA class P molecule disclosed herein.
  • the method comprises contacting the T cells with an APC disclosed herein.
  • the enriched population of T cells comprises a higher number of T cells capable of binding the HLA class P molecule relative to the number of T cells capable of binding the HLA class P molecule prior to the contacting.
  • the method comprises contacting the T cells in vitro with a peptide, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the method comprises contacting the T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, following the contacting, the enriched population of T cells comprises a higher number of T cells capable of binding the HLA class P molecule relative to the number of T cells capable of binding the HLA class P molecule prior to the contacting.
  • Some aspects of the present disclosure are directed to a method of selecting a T cell capable of targeting a tumor cell.
  • the method comprises contacting a population of isolated T cells in vitro with a peptide, wherein the peptide consists of an amino acid sequence as set forth in SEQ ID NO: 13.
  • the T cells are obtained from a human subject.
  • the T cells obtained from the human subject can be any T cells disclosed herein.
  • the T cells obtained from the human subject are tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the method further comprises administering to the human subject the enriched T cells.
  • the subject is preconditioned prior to receiving the T cells, as described herein.
  • Peripheral mononuclear cells were obtained via density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare Life Sciences, Marlborough, MA).
  • the K562 cell line is an erythroleukemic cell line with defective HLA class I/P expression.
  • K562-based artificial APCs aAPCs individually expressing various HLA class P genes as a single HLA allele in conjunction with CD80 and CD83 have been reported previously (Butler et al., PloS One 7, e30229 (2012).
  • the Jurkat 76 cell line is a T cell leukemic cell line lacking endogenous TCR, CD4, and CDS expression.
  • Jurkat 76/CD4 cells were generated by retrovirally transducing the human CD4 gene.
  • HEK293T cells and melanoma cell lines were grown in DMEM supplemented with 10% FBS and 50 pg/ml gentamicin (Thermo Fisher Scientific, Waltham,
  • the K562 and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 mg/ml gentamicin.
  • the CCND 1219-238 synthetic peptide was purchased from Genscript (Piscataway, NJ) and dissolved at 50 mg/ml in DMSO.
  • Novel TCR genes were cloned via 5’-rapid amplification of cDNA ends (RACE) PCR using SMARTer RACE 573’ Kit (Takara Bio, Shiga, Japan) and sequenced as previously described. All genes were cloned into the pMX retroviral vector and transduced into cell lines using the 293GPG and PG13 cell-based retrovirus system.
  • APC-Cy7- conjugated anti-CD4 RPA-T4, Biolegend, San Diego, CA 44
  • PE-conjugated anti-His tag ADI.1.10, Abeam, Cambridge, MA
  • Dead cells were distinguished with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit 465 (Thermo Fisher Scientific, Waltham, MA).
  • Stained cells were analyzed with Canto P or LSRFortessa X-20 (BD Biosciences, Franklin Lakes, NJ). Cell sorting was conducted using a FACS Aria P (BD Biosciences, Franklin Lakes, NJ). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).
  • anti ⁇ -actin C4, Santa Cruz Biotechnology, Santa Cruz, CA
  • anti-CCNDl EPR2241, Abeam, Cambridge, MA
  • HRP-conjugated goat anti-mouse IgG H+L secondary antibody
  • HRP-conjugated anti-rabbit IgG H+L secondary antibody
  • CD3 + and CD4 + T cells were purified using the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD4 + T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Purified T cells were stimulated with aAPC/mOKT3 irradiated with 200 Gy at an E:T ratio of 20:1. Starting the following day, activated T cells were retrovirally transduced with the cloned TCR genes via centrifugation for 1 hour at 1,000 x g at 32°C for 3 consecutive days or using a Retronectin-coated plate (Takara Bio, Shiga, Japan). On the following day, 100 IU/ml IL-2 and 10 ng/ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2-3 days.
  • the extracellular domain of the wild-type class II a gene was fused with an acidic leucine zipper via a GGGS linker followed by a 6xHis tag via a GS linker ⁇ see SEQ ID NO: 15; Table 5).
  • the ectodomain of the class P b gene carrying mutations ⁇ see SEQ ID NO: 14) was similarly linked with a basic leucine zipper via a GGGS linker ⁇ see SEQ ID NO: 14).
  • HEK293T cells and A375 cells were transfected with the a and b genes using the 293GPG cell-based retrovirus system and cultured in DMEM supplemented with 10% FBS and 50 mg/ml gentamicin.
  • HEK293T cells stably secreting soluble DP4 L112W/V141M protein were grown until confluent, and the medium was changed to serum- free 293 SFM P medium (Thermo Fisher Scientific, Waltham, MA). After forty-eight hours, the conditioned medium was harvested and concentrated using Ami con Ultra filters (molecular weight cut-off (MWCO) 10 kDa) (MilliporeSigma, Burlington, MA). The soluble HLA class II-containing supernatant was then mixed with 100 mg/ml peptide of interest for 20-24 hours at 37°C for in vitro peptide exchange. Monomer that was not subjected to peptide exchange was used as a control.
  • MWCO molecular weight cut-off
  • the concentration of the monomer was measured by specific ELISA using a nickel-coated plate (XPressBio, Frederick, MD) and an anti-His tag biotinylated mAb (ADI.1.10, R&D Systems, Minneapolis, MN). Soluble HLA class P monomer was dimerized using PE-conjugated anti-His mAb (AD 1.1.10, Abeam, Cambridge, MA) at a 2: 1 molar ratio for 1.5 hours at 4°C for staining.
  • CD4 + T cells were purified using a CD4 + T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified T cells were stimulated with DP4-expressing aAPCs pulsed with DP4-restricted peptides at 10 mg/ml and irradiated at 200 Gy at an E:T ratio of 20:1. After forty-eight hours, 10 IU/ml IL-2 and 10 ng/ml IL-15 were added to the CD4 + T cells. The culture medium supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) was replenished every 2-3 days. After 2 weeks of stimulation, the T cells were subjected to DP4
  • Cytokine ELISPOT assays were performed as previously reported (see, e.g., Yamashita et al., Nat. Commun. 8:15244 (2017); and Anczurowski et al., Sci. Rep. 8:4804 (2018)).
  • the TCR 03 -CCND1219-238 was able to recognize a cognate peptide that was endogenously processed and presented by DP4 (FIGs. 4A-4E and 5A-5B).

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