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WO2023215302A2 - Polythérapies comprenant des coupleurs d'antigènes de lymphocytes t et des inhibiteurs de points de contrôle pour le traitement du cancer - Google Patents

Polythérapies comprenant des coupleurs d'antigènes de lymphocytes t et des inhibiteurs de points de contrôle pour le traitement du cancer Download PDF

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Publication number
WO2023215302A2
WO2023215302A2 PCT/US2023/020709 US2023020709W WO2023215302A2 WO 2023215302 A2 WO2023215302 A2 WO 2023215302A2 US 2023020709 W US2023020709 W US 2023020709W WO 2023215302 A2 WO2023215302 A2 WO 2023215302A2
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Prior art keywords
antigen
seq
cell
amino acid
binding domain
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PCT/US2023/020709
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WO2023215302A3 (fr
Inventor
Christopher W. HELSEN
Sadhak SENGUPTA
Heather MACGREGOR
Philbert IP
Andreas Bader
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Triumvira Immunologics Usa, Inc.
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Publication of WO2023215302A2 publication Critical patent/WO2023215302A2/fr
Publication of WO2023215302A3 publication Critical patent/WO2023215302A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4637—Other peptides or polypeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • a cancer expressing a target antigen in a subject in need thereof comprising administering to the subject: (a) an engineered T cell comprising an expression vector encoding a T cell-antigen coupler (TAC) protein: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR co-receptor, wherein the antigen-binding domain, the ligand that binds a protein associated with TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other or joined by at least one linker; and (b) an immune checkpoint inhibitor.
  • TAC T cell-antigen coupler
  • the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently.
  • the target antigen is human epidermal grow th factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3). In some embodiments, the target antigen is HER2. In some embodiments, the antigen-binding domain is a designed ankyrin repeat protein (DARPin).
  • DARPin ankyrin repeat protein
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49.
  • the target antigen is BCMA.
  • the antigen-binding domain is a single-cham variable fragment (scFv).
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50.
  • the target antigen is CD19.
  • the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the target antigen is Claudin 18.2 (CLDN18.2). In some embodiments, the antigen-binding domain is a nanobody.
  • the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53.
  • the target antigen is GUCY2C.
  • the antigen-binding domain is an scFv.
  • the antigen-bmdmg domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54- 117.
  • the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136. In some embodiments, the target antigen is GPC3. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137.
  • the ligand that binds a protein associated with a TCR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain and comprises a threonine (T) residue at amino acid position 177.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.
  • the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigenbinding domain.
  • the ligand that binds a protein associated with a TCR complex compnses an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.
  • the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.
  • the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.
  • the TCR co-receptor is CD4. In some embodiments, the TCR co-receptor is CD8, preferably CD8a. In some embodiments, the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused. In some embodiments, the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker.
  • the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker.
  • the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 amino acids, or 15 amino acids.
  • the peptide linker comprises a GiSi linker.
  • the TAC does not comprise a co-stimulatory domain. In some embodiments, the TAC does not comprise an activation domain.
  • the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207.
  • the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor.
  • the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is ipilimumab or tremelimumab. In some embodiments, the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable.
  • the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer.
  • the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma.
  • the subject has received two or more prior lines of therapy.
  • the engineered T cells are autologous to the subject.
  • the engineered T cells are heterologous to the subject.
  • the engineered T cells are administered in a single dose.
  • the engineered T cells are administered at a dose of about 6* 10 4 to about 6x 10 s cells/kg body weight.
  • the engineered T cells are administered at a dose of about IxlO 5 to about 8x l0 6 cells/kg body weight.
  • the immune checkpoint inhibitor is administered at a dose of 200 mg.
  • the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered.
  • the immune checkpoint inhibitor is administered once every three weeks.
  • TAC T cell-antigen coupler
  • the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently.
  • an engineered T cell comprising an expression vector encoding a T cell-antigen coupler (TAC) protein and an immune checkpoint inhibitor in combination for the manufacture of a medicament for treating a cancer expressing a target antigen in a subject in need thereof, comprising administering to the subject: (a) the engineered T cell comprising the expression vector encoding the T cell-antigen coupler (TAC) protein, the TAC protein comprising: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR coreceptor, wherein the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR coreceptor, wherein the antigen-binding domain,
  • the TAC protein is administered prior to administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein is administered following administration of the immune checkpoint inhibitor. In some embodiments, the TAC protein and the immune checkpoint inhibitor are administered concurrently.
  • the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3). In some embodiments, the target antigen is HER2. In some embodiments, the antigen-binding domain is a designed ankyrin repeat protein (DARPin).
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49.
  • the target antigen is BCMA.
  • the antigen-binding domain is a single-chain variable fragment (scFv).
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50.
  • the target antigen is CD19.
  • the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the target antigen is Claudin 18.2 (CLDN18.2). In some embodiments, the antigen-binding domain is a nanobody.
  • the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53.
  • the target antigen is GUCY2C.
  • the antigen-binding domain is an scFv.
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54- 117.
  • the antigen-binding domain is a nanobody. In some embodiments, the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136. In some embodiments, the target antigen is GPC3. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigenbinding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137.
  • the ligand that binds a protein associated with a TOR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K.
  • the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain and comprises a threonine (T) residue at amino acid position 177.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.
  • the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigen- binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.
  • the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.
  • the TCR co-receptor is CD4.
  • the TCR co-receptor is CD8, preferably CD8a.
  • the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused.
  • the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker.
  • the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker.
  • the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 amino acids, or 15 ammo acids.
  • the peptide linker comprises a G4S3 linker.
  • the TAC does not comprise a co-stimulatory domain.
  • the TAC does not comprise an activation domain.
  • the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207.
  • the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor.
  • the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is ipilimumab or tremelimumab.
  • the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable In some embodiments, the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer.
  • the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma.
  • the subject has received two or more prior lines of therapy.
  • the engineered T cells are autologous to the subject.
  • the engineered T cells are heterologous to the subject.
  • the engineered T cells are administered in a single dose.
  • the engineered T cells are administered at a dose of about 6x IO 4 to about 6x l0 8 cells/kg body weight.
  • the engineered T cells are administered at a dose of about IxlO 5 to about 8x l0 6 cells/kg body weight.
  • the immune checkpoint inhibitor is administered at a dose of 200 mg.
  • the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered.
  • the immune checkpoint inhibitor is administered once every three weeks.
  • TAC T cell-antigen coupler
  • the TAC protein comprising: (i) an antigen-binding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (in) a transmembrane domain and a cytosolic domain of a TCR co-receptor; wherein the antigenbinding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other, or joined by at least one linker; for use in the treatment of cancer expressing a target antigen in a subject in need thereof, wherein the T cell-antigen coupler (TAC) protein is administered to the individual in combination with an immune checkpoint inhibitor.
  • TAC T cell-antigen coupler
  • TAC T cell-antigen coupler
  • the TAC protein comprising: (i) an antigenbinding domain that binds the target antigen; (ii) a ligand that binds a protein associated with a T cell receptor (TCR) complex on the engineered T cell; and (iii) a transmembrane domain and a cytosolic domain of a TCR co-receptor; wherein the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are fused directly to each other, or joined by at least one linker, for use in the treatment of cancer expressing a tumor antigen in an individual in need thereof, wherein the engineered T cell comprising an expression vector encoding a TAC protein is administered simultaneously, separately or sequentially with an immune check
  • the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin 18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3).
  • the target antigen is HER2.
  • the antigen-binding domain is a designed ankyrin repeat protein (DARPin).
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 49.
  • the target antigen is BCMA.
  • the antigen-binding domain is a single-chain variable fragment (scFv).
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 50.
  • the target antigen is CD 19.
  • the antigen-binding domain is an scFv.
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 51.
  • the target antigen is Claudin 18.2 (CLDN18.2).
  • the antigen-binding domain is a nanobody.
  • the antigen-binding domain comprises an ammo acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52 or 53.
  • the target antigen is GUCY2C.
  • the antigen-binding domain is an scFv.
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 54-117.
  • the antigen-binding domain is a nanobody.
  • the antigen-binding domain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 118-136.
  • the target antigen is GPC3.
  • the antigen-binding domain is an scFv.
  • the antigen-binding domain comprises an ammo acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137.
  • the ligand that binds a protein associated with a TCR complex is an scFv derived from an antigen-binding domain selected from UCHT1, huUCHTl, OKT3, F6A, and L2K. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2.
  • the ligand that binds a protein associated with a TCR complex is derived from a UCHT1 antigenbinding domain and comprises a threonine (T) residue at amino acid position 177.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.
  • the ligand that binds a protein associated with a TCR complex is derived from an huUCHTl antigen-binding domain and comprises a threonine (T) residue at amino acid position 182.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.
  • the ligand that binds a protein associated with a TCR complex is derived from an OKT3 antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.
  • the ligand that binds a protein associated with a TCR complex is derived from an F6A antigen-binding domain. In some embodiments, the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the ligand that binds a protein associated with a TCR complex is derived from an L2K antigen-binding domain.
  • the ligand that binds a protein associated with a TCR complex comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.
  • the TCR co-receptor is CD4.
  • the TCR co-receptor is CD8, preferably CD8a.
  • the antigen-binding domain, the ligand that binds a protein associated with a TCR complex, and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused.
  • the antigen-binding domain and the ligand that binds a protein associated with a TCR complex are directly fused and joined to the transmembrane domain and cytosolic domain of a TCR co-receptor by a linker.
  • the ligand that binds a protein associated with a TCR complex and the transmembrane domain and cytosolic domain of a TCR co-receptor are directly fused and joined to the antigen-binding domain by a linker.
  • the linker is a peptide linker, preferably a peptide linker comprising 5 to 30 amino acids, more preferably 5 amino acids, 10 ammo acids, or 15 amino acids.
  • the peptide linker comprises a G4S3 linker.
  • the TAC does not comprise a co-stimulatory domain.
  • the TAC does not comprise an activation domain.
  • the TAC comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 138-207.
  • the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor.
  • the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezohzumab, avelumab, durvalumab.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is ipilimumab or tremelimumab.
  • the cancer is a solid or liquid cancer. In some embodiments, the cancer is a primary or metastatic cancer. In some embodiments, the cancer is unresectable.
  • the cancer is a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer, a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, an endometrial cancer.
  • the cancer is an acute lymphoblastic leukemia, a chronic lymphocytic leukemia, a large B-cell lymphoma, or a diffuse large B-cell lymphoma.
  • the subject has received two or more prior lines of therapy.
  • the engineered T cells are autologous to the subject.
  • the engineered T cells are heterologous to the subject.
  • the engineered T cells are administered in a single dose.
  • the engineered T cells are administered at a dose of about 6x 10 4 to about 6x 10 s cells/kg body weight.
  • the engineered T cells are administered at a dose of about lx JO 5 to about 8x l0 6 cells/kg body weight.
  • the immune checkpoint inhibitor is administered at a dose of 200 mg.
  • the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered.
  • the immune checkpoint inhibitor is administered once every three weeks.
  • FIGs. 1A-1B depict an in vivo experiment assessing the effects of HER2-TAC T cells on HER + xenograft tumors in mice.
  • FIG. 1A diagrams the design of the experiment.
  • FIG. IB is a graph depicting tumor volumes measured over the course of the experiment.
  • FIG. 1C depicts measurement of CD69 (vertical axis) and PD-1 (horizonal axis) in cells collected from xenografted mice sacrificed 30 minutes after engraftment of HER2-TAC T cells.
  • FIG. ID depicts measurement of CD69 (vertical axis) and PD-1 (horizonal axis) in cells collected from xenografted mice sacrificed 7 days after engraftment of HER2-TAC T cells.
  • FIGs. 2A-2C depict an in vitro experiment measuring the effects of HER2-TAC T cells on HER2 + tumor spheroids engineered to express green fluorescent protein (GFP).
  • FIG. 2A depicts fluorescence images of spheroids treated with HER2-TAC T cells or controls at indicated effector target (E:T) ratios.
  • FIG. 2B depicts percent viability of tumor cells in indicated treatment groups after 4 days of treatment, as measured by normalized GFP fluorescence.
  • 2C depicts images of tumor spheroids treated with HER2-TAC T cells (top panels) or control (bottom panels) that were fixed and stained for (from left-to-right) hematoxylin and eosin (HE), CD3, granzyme B, PD-L1, HER2, and cleaved caspase 3.
  • HE hematoxylin and eosin
  • FIGs. 3A-3C depict an experiment assessing the effects of anti-PD-1 treatment on TAC T cells activated against PD-L1 overexpressing tumor cells.
  • FIG. 3A depicts flow cytometry measurement of surface expression of PD-L1 in BT-474 cells engineered to overexpress PD-L1.
  • FIG. 3B depicts flow cytometry measurement of surface expression of PD-L1 in N87 cells engineered to overexpress PD-L1.
  • FIG. 3C is a graph showing proliferation of HER2-TAC T cells as assessed by CTV dye staining after exposure to indicated HER2 + cell lines with and without pembrolizumab co-treatment.
  • FIGs. 4A-4B depict an in vitro experiment assessing the effects of HER2-TAC T cell treatment, administered with or without pembrolizumab, on HER2-expressing N87 WT/nuc GFP or N87 PD ' L1 ' IIlsh/nuc GFP cancer cells.
  • FIG. 4A depicts cell images taken at the experimental endpoint (i.e., day 6, hour 144), with live tumor cells shown in green and death dye DRAQ7 shown in red.
  • FIG. 4B is a graph showing the percentage of live cells quantified at each timepoint of the experiment (i.e., every 8 hours, up until hour 144).
  • FIG. 5 depicts the design of a clinical trial assessing a HER2-TAC T cell + pembrolizumab combination therapy for treating cancer patients.
  • TAC antigen-specific T cell-antigen coupler
  • T cells can be genetically modified to yield: (i) forced expression of T cell receptor (TCR); or (ii) a chimeric antigen receptor (CAR) specific for antigen targets on the tumor.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the chimeric antigen receptors used for engineering T cells consist of: (i) a targeting domain, usually a singlechain fragment variable (scFv); (ii) a transmembrane domain; and (iii) a cytosolic domain that contains signaling elements from the T cell receptor and associated proteins.
  • a targeting domain usually a singlechain fragment variable (scFv);
  • a transmembrane domain usually a singlechain fragment variable
  • cytosolic domain that contains signaling elements from the T cell receptor and associated proteins.
  • Such chimeric antigen receptors have also been referred to as “T-body” or “Chimeric Immune Receptor” (CIR), but currently, most researchers use the term “CAR”.
  • CAR CAR
  • One advantage of the CAR approach is that it allows any patient’s immune cells to be targeted against any desirable target in a major histocompatibility complex (MHC) independent manner. This is appealing as MHC presentation is often defective in tumor cells.
  • MHC major histocomp
  • CAR-engineered T cells have shown considerable promise in clinical application, they rely on a synthetic method for replacing the native activation signal that is provided by the T cell receptor (TCR). Furthermore, since the CAR signaling domains are disconnected from their natural regulatory partners by the very nature of the CAR structure, there is an inherent risk that CARs may lead to a low-level of constitutive activation, which could result in off-target toxicities. Given these limitations, it is preferable to re-direct T cells to attack tumors via their natural TCR. To this end, a class of recombinant proteins termed “Bispecific T-cell Engagers” (BiTEs) has been created. These proteins employ bispecific antibody fragments to crosslink T- cell TCR receptors with target antigens.
  • BiTEs Bispecific T-cell Engagers
  • bi-specific antibodies have been generated that accomplish this goal and some scientists have simply linked anti-CD3 antibodies to tumor-specific antibodies employing chemical linkage. While these bi-specific proteins have demonstrated some activity in vitro, GMP production, short biological half-lives, and limited bioavailability represent significant challenges to the successful use of these molecules in cancer treatment. Additionally, these molecules also fail to properly recapitulate natural TCR signaling because they do not engage the TCR co-receptors (CD8 and CD4).
  • TAC T cell Antigen Coupler
  • TAC T cell Antigen Coupler
  • TAC T cell Antigen Coupler
  • TACs disclosed herein activate natural Major Histocompatibility complex (MHC) signaling through the T-cell receptor (TCR), while retaining MHC -unrestricted targeting.
  • MHC Major Histocompatibility complex
  • TACs disclosed herein recruit the T-Cell Receptor (TCR) in combination with co-receptor stimulation.
  • TACs disclosed herein show enhanced activity and safety.
  • antigen-binding domain refers to any substance or molecule that binds, directly or indirectly, to a target (e.g, Claudin 18.2).
  • Antigen-binding domains include antibodies or fragments thereof, peptides, peptidomimetics, proteins, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, or small molecules that bind to a target.
  • antibody is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g, an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated.
  • antibodies are multimeric proteins that contain four polypeptide chains.
  • immunoglobulin heavy chains H chains
  • immunoglobulin light chains L chains
  • the immunoglobulin heavy and light chains are connected by an interchain disulfide bond.
  • the immunoglobulin heavy chains are connected by interchain disulfide bonds.
  • a light chain consists of one variable region (VL) and one constant region (CL).
  • the heavy chain consists of one variable region (VH) and at least three constant regions (CHI, CH2 and CH3).
  • the variable regions determine the binding specificity of the antibody.
  • Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs).
  • CDRs complementarity determining regions
  • antibody-based antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.
  • antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g, bispecific antibodies).
  • An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
  • nucleic acid sequence refers to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurnng monomers or portions thereof.
  • the nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil The sequences may also contain modified bases.
  • modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.
  • the nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination or obtained by chemical synthesis or other known protocols for creating nucleic acids.
  • recombinant nucleic acid or “engineered nucleic acid” as used herein refers to a nucleic acid or polynucleotide that is not found in a biological organism.
  • recombinant nucleic acids may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create sequences that would not otherwise be found in nature.
  • Recombinant nucleic acids may also be created by chemical synthesis or other known protocols for creating nucleic acids.
  • peptide means a chain of amino acids.
  • protein as used herein further means a large molecule comprising one or more chains of amino acids and, in some embodiments, is a fragment or domain of a protein or a full- length protein.
  • protein either refers to a linear chain of amino acids or to a chain of amino acids that has been processed and folded into a functional protein.
  • the protein structure is divided into four distinct levels: (1) primary structure - referring to the sequence of amino acids in the polypeptide chain, (2) secondary structure - referring to the regular local sub-structures on the polypeptide backbone chain, such as a-helix and -sheets, (3) tertiary structure - referring to the three-dimensional structure if monomeric and multimeric protein molecules, and (4) quaternary structure - referring to the three-dimensional structure comprising the aggregation of two or more individual polypeptide chains that operate as a single functional unit.
  • the use of peptide or polypeptide herein does not mean that the chain of amino acids is not also a protein (i.e., a chain of amino acids having a secondary , tertiary or quaternary structure).
  • a vector refers to a polynucleotide that is used to deliver a nucleic acid to the inside of a cell.
  • a vector is an expression vector comprising expression control sequences (for example, a promoter) operatively linked to a nucleic acid to be expressed in a cell.
  • Expression control sequences for example, a promoter
  • Vectors known in the art include, but are not limited to, plasmids, phages, cosmids and viruses.
  • tumor antigen or “tumor associated antigen” as used herein refers to a substance produced by tumor cells, which is recognized by the immune system, and which elicits an antigen-specific immune response in a host (e.g., which is presented by MHC complexes).
  • a tumor antigen is on the surface of a tumor cell.
  • transmembrane and cytosolic domain refers to a polypeptide that comprises a transmembrane domain and a cytosolic domain of a protein associated with the T cell receptor (TCR) complex.
  • TCR T cell receptor
  • such transmembrane and cytosolic domain may include, but is not limited to, protein domains that (a) associate with the lipid raft and/or (b) bind Lek.
  • TCR co-receptor refers to a molecule that assists the T cell receptor (TCR) in communicating with an antigen-presenting cell.
  • TCR co-receptors include, but are not limited to, CD4, LAG3, and CD8.
  • TCR co-stimulators include, but are not limited to, ICOS, CD27, CD28, 4-1BB (CD 137), 0X40 (CD134), CD30, CD40, lymphocyte fiction- associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.
  • the terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.
  • treatment refers to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of affecting a partial or complete cure for a disease and/or symptoms of the disease.
  • Treatment may include treatment of a disease or disorder (e.g.
  • cancer in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g, including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician.
  • the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., cancer).
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • singular forms “a”, “and,” and “the” include plural referents unless the context clearly indicates otherwise.
  • reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.
  • reference to a range of 1-5,000-fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.
  • “About” a number refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.
  • Percent (%) identity refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment.
  • an amino acid sequence is X% identical to SEQ ID NO: Y refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y.
  • computer programs are employed for such calculations.
  • Exemplary programs that compare and align pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).
  • selective binding refers to the higher affinity with which a molecule (e.g., protein such as an antigen-binding domain of TAC) binds its target molecule (e.g, target antigen such as HER2) over other molecules.
  • a molecule e.g., protein such as an antigen-binding domain of TAC
  • target molecule e.g, target antigen such as HER2
  • selective binding and “specific binding” are used interchangeably herein.
  • a target antigen-binding domain of a TAC polypeptide disclosed herein specifically binds to a target antigen on a tumor cell.
  • a target antigen in a subject in need thereof, comprising administering to the individual an engineered TAC T cell that targets said antigen in combination with an immune checkpoint inhibitor, for example a checkpoint inhibitor disclosed herein.
  • an immune checkpoint inhibitor for example a checkpoint inhibitor disclosed herein.
  • the TAC protein is administered prior to administration of the immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is administered 7 days after the engineered T cells are administered.
  • the immune checkpoint inhibitor is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 days after the engineered T cells are administered.
  • the immune checkpoint inhibitor is administered 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more than 8 weeks after the engineered T cells are administered.
  • the TAC protein is administered following administration of the immune checkpoint inhibitor.
  • the TAC protein and the immune checkpoint inhibitor are administered concurrently.
  • the methods further comprise administering a lymphodepl eting therapy, or are administered to a subject who has received a lymphodepleting therapy.
  • lymphodepl eting therapies include nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.
  • TBI total body irradiation
  • cancers that are treated include, but are not limited to, a salivary gland cancer, a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer (e.g, an endometrial cancer), a cervical cancer, a biliary tract cancer, a pancreatic cancer, a colorectal cancer, a bladder cancer (e.g., a urothelial cancer), a prostate cancer, multiple myeloma, a glioblastoma, a gastroesophageal junction cancer, an esophageal cancer, a liver cancer, a thyroid cancer, a kidney cancer, a yolk sac tumor, a skin cancer, or a sarcoma.
  • a salivary gland cancer e.g., a lung cancer, a gastric cancer, a breast cancer, an ovarian cancer, a uterine cancer (e.g, an endometrial cancer), a cervical cancer, a biliary tract cancer,
  • the cancer is breast, lung, pancreatic, colorectal, gastric, endometrial, or ovarian cancer. In some embodiments, the cancer is rectosigmoid cancer, gastro-esophageal cancer, or gastric adenocarcinoma.
  • the cancer is a liver cancer (for example, HCC), gastric carcinoma, ovarian carcinoma (for example, ovarian clear cell carcinoma), melanoma, colorectal carcinoma, thyroid cancer, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma, or yolk sac tumor.
  • HCC liver cancer
  • gastric carcinoma for example, gastric carcinoma
  • ovarian carcinoma for example, ovarian clear cell carcinoma
  • melanoma for example, ovarian clear cell carcinoma
  • colorectal carcinoma for example, thyroid cancer
  • squamous cell carcinoma of the lung hepatoblastoma, nephroblastoma, or yolk sac tumor.
  • cancers that are treated include, but are not limited to, a pancreatic cancer (e.g., pancreatic adenocarcinoma, a gastric cancer (e.g, gastric adenocarcinoma), a gastroesophageal cancer (e.g., gastroesophageal junction (GEJ) adenocarcinoma), an esophageal cancer, an ovarian cancer, or a lung cancer (e.g., non-small cell lung cancer).
  • the cancer is a primary colorectal cancer, a primary gastric cancer, a primary gastroesophageal junction cancer, a primary esophageal cancer, or a primary pancreatic cancer.
  • the cancer is a metastatic colorectal cancer, a metastatic gastric cancer, a metastatic gastroesophageal junction cancer, a metastatic esophageal cancer, or a metastatic pancreatic cancer.
  • cancers that are treated include, but are not limited to, multiple myeloma, B cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or Non-Hodgkin’s Lymphoma.
  • the cancer to be treated is non-Hodgkin’s lymphoma, such as a B-cell lymphoma.
  • the non-Hodgkin’s lymphoma is a B-cell lymphoma, such as a large B-cell lymphoma (LBCL), a diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, high grade B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL), or primary central nervous system lymphoma.
  • TAC T cell-antigen coupler
  • the methods disclosed herein employ a T cell-antigen coupler (TAC) polypeptide.
  • TAC polypeptide includes a target antigenbinding domain, which binds to a target antigen of interest (e.g., an antigen expressed on a cancer cell); a ligand that binds a protein associated with a T cell receptor (TCR) (e.g, a CD3 protein, e.g., CD3E); and a transmembrane and cytosolic domain of a TCR co-receptor (e.g, CD4 or CD8).
  • TCR T cell receptor
  • the target antigen-binding domain can allow the T cell to specifically bind to a cell expressing the target antigen (e.g., a cancer cell).
  • the ligand that binds a protein associated with a TCR can localize the TAC to the TCR on a T cell.
  • the transmembrane and cytosolic domain of a TCR co-receptor can allow T cell activation through endogenous TCR signaling by inducing Lck-mediated phosphorylation of immunotyrosine activation motifs (ITAMs) on TCRs.
  • the TAC polypeptide does not include a co-stimulatory domain.
  • the TAC polypeptide does not include a co-activation domain.
  • the TAC comprises an antigen-binding domain that binds a protein associated with the TCR complex.
  • a “TCR complex protein antigen-binding domain,” also referred to as a “TCR complex antigen-binding domain,” “antigen-binding domain that binds the TCR complex,” or “antigen-binding domain that binds a protein associated with the TCR complex,” refers to any substance or molecule that binds, directly or indirectly, toa protein associated with a TCR complex.
  • the antigen-binding domain that binds a protein associated with a TCR complex selectively binds to a protein of the TCR.
  • the antigen-binding domain that binds a protein associated with a TCR complex comprises a substance that specifically binds to a protein of the TCR.
  • the TCR complex protein antigen-binding domain is selected from antibodies or fragments thereof, for example, single chain antibodies (e.g, single-chain fragment variable antibodies (scFvs)), single domain antibodies (e.g., nanobodies (VHH), shark heavy-chain-only antibodies (VNAR)), diabodies, minibodies, Fab fragments, Fab' fragments, F(ab’)2 fragments, or Fv fragments that bind to a protein of the TCR.
  • single chain antibodies e.g, single-chain fragment variable antibodies (scFvs)
  • single domain antibodies e.g., nanobodies (VHH), shark heavy-chain-only antibodies (VNAR)
  • diabodies minibodies
  • Fab fragments Fab' fragments
  • F(ab’)2 fragments fragments that bind to a protein of the TCR.
  • the TCR complex protein antigen-binding domain is selected from ankyrin repeat proteins (DARPins), affibodies, adnectins, affilins, phylomers: fynomers, affimers, peptide aptamers, lectins, knottins, centyrins, anticalins, peptides, peptidomimetics, proteins, glycoproteins, or proteoglycans that bind to a protein of the TCR, or naturally occurring ligands for a protein of the TCR.
  • DARPins ankyrin repeat proteins
  • the TCR complex protein antigen-binding domain is a nonprotein compound that binds to a protein of the TCR, including but not limited to carbohydrates, lipids, nucleic acids, or small molecules.
  • the TCR complex protein antigen-binding domain is a designed ankyrin repeat (DARPin) targeted to a protein of the TCR.
  • the TCR complex protein antigen-binding domain is a single-chain variable fragment (ScFv) targeted to a protein of the TCR.
  • the TCR complex protein antigen-binding domain is a nanobody targeted to a protein of the TCR.
  • Proteins associated with the TCR include, but are not limited, to the TCR alpha (a) chain, TCR beta (P) chain, TCR gamma (y) chain, TCR delta (5) chain, CD3y chain, CD33 chain and CD3e chains.
  • an antigen-binding domain that binds a protein associated with the TCR complex is an antibody to the TCR alpha (a) chain, TCR beta ( ) chain, TCR gamma (y) chain, TCR delta (3) chain, CD3y chain, CD33 chain and/or CD3E chain.
  • the protein associated with a TCR complex is CD3.
  • the protein associated with a TCR complex is CD3E.
  • the antigen-binding domain that binds CD3 is an antibody, for example, a single chain antibody, for example a single-chain variable fragment (scFv).
  • CD3 antibodies include, but are not limited to, UCHT1, OKT3, F6A, L2K, muromonab, otelixizumab, teplizumab, visilizumab, CD3-12, MEM-57, 4D10A6, CD3D, or TR66.
  • the antigen-binding domain that binds the TCR complex is UCHT1, or a variant thereof.
  • the UCHT1 antigen-binding domain is encoded by SEQ ID NO: 1.
  • the UCHT1 antigen-binding domain comprises SEQ ID NO: 2.
  • the UCHT1 antigen-binding domain is mutated.
  • the UCHT1 antigen-binding domain comprises a Y to T mutation at a position corresponding to amino acid 182 of SEQ ID NO: 32 (Y182T).
  • the UCHT1 (Y182T) antigen-binding domain is encoded by SEQ ID NO: 3.
  • the UCHT1 (Y182T) antigen-binding domain comprises SEQ ID NO: 4.
  • the antigen-binding domain that binds the TCR complex is a humanized UCHT1 (huUCHTl).
  • the huUCHTl antigen-binding domain is encoded by SEQ ID NO: 5.
  • the huUCHTl antigen-binding domain comprises SEQ ID NO: 6.
  • the huUCHTl has a Y to T mutation at a position corresponding to amino acid 177 of SEQ ID NO: 6 (Y177T).
  • the huUCHTl (Y177T) antigen-binding domain is encoded by SEQ ID NO: 7.
  • the huUCHTl antigen-binding domain comprises SEQ ID NO: 8.
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 1 (UCHT1).
  • the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCEIT1).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1 ). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex compnses a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 1 (UCHT1). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 1 (UCHT1).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity' with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 2 (UCHT1). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1) (z.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g., framew ork) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the non-CDR e.g., framew ork sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e .g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g., framew ork) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the non-CDR e.g., framew ork sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 2 (UCHT1).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 3 (UCHT1 (Y182T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity' with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen- binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)). In some embodiments, the antigen- binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non- CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 4 (UCHT1 (Y182T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 97% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 5 (huUCHTl). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 5 (huUCHTl).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the ammo acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 98% sequence identity' with the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the ammo acid sequence of SEQ ID NO: 6 (huUCHTl). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e g., framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g. , framework) sequences of the amino acid sequence of SEQ ID NO: 6 (huUCHTl).
  • the polynucleotide encoding the antigen-bindmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex compnses a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 7 (huUCHTl (Y177T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the antigen-binding domain that binds the protein associated wi th the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigenbinding domain that binds the protein associated with the TCR complex have at least 80% sequence identity' with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 85% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framew ork) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 90% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 95% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g, framework) sequences of the antigenbinding domain that binds the protein associated with the TCR complex have at least 96% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigenbinding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 97% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framew ork) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 98% sequence identity with the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 99% sequence identity with the non-CDR (e.g, framework) sequences of the amino acid sequence of SEQ ID NO: 8 (huUCHTl (Y177T)).
  • the antigen-binding domain that binds to the protein associated with the TCR complex is OKT3.
  • the murine OKT3 antigen-binding domain is encoded by SEQ ID NO: 9.
  • the OKT3 antigen-binding domain comprises SEQ ID NO: 10.
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 9 (OKT3).
  • the polynucleotide encoding the antigen-bmdmg domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 9 (OKT3). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 9 (OKT3). [0056] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 10 (OKT3).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity' with the amino acid sequence of SEQ ID NO: 10 (OKT3). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 10 (OKT3).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 10 (OKT3) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 10 (OKT3).
  • the antigen-binding domain that binds to the protein associated with the TCR complex is F6A.
  • the murine F6A antigen-binding domain is encoded by SEQ ID NO: 11.
  • the F6A antigen-binding domain comprises SEQ ID NO: 12.
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 11 (F6A).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 11 (F6A). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 11 (F6A). [0060] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity' with the amino acid sequence of SEQ ID NO: 12 (F6A).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 12 (F6A). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 12 (F6A).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 12 (F6A) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 12 (F6A).
  • the antigen-binding domain that binds to the protein associated with the TCR complex is L2K.
  • the murine L2K antigen-binding domain is encoded by SEQ ID NO: 13.
  • the L2K antigen-binding domain comprises SEQ ID NO: 14.
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 80% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 90% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 95% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K).
  • the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 13 (L2K). In some embodiments, the polynucleotide encoding the antigen-binding domain that binds the protein associated with the TCR complex comprises the nucleotide sequence of SEQ ID NO: 13 (L2K). [0064] In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K).
  • the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigenbinding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K).
  • the antigen-binding domain that binds the protein associated with the TCR complex compnses an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 14 (L2K). In some embodiments, the antigen-binding domain that binds the protein associated with the TCR complex comprises the amino acid sequence of SEQ ID NO: 14 (L2K).
  • the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 14 (L2K) (i.e., the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each having 100% identity to the corresponding CDR in the amino acid sequence of SEQ ID NO: 14 (L2K).
  • Table 1 Table of Sequences 1 Light chain, nucleotides 1-324; Linker, nucleotides 325-387; Heavy chain, nucleotides 388- 750
  • a nucleic acid disclosed herein is in an order of (1) a polynucleotide encoding a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain.
  • a UCHT1 e.g., huUCHTl
  • CD4 transmembrane domain e.g., CD4 transmembrane domain
  • a nucleic acid disclosed herein is in an order of (1) a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 5’ end to 3’ end.
  • a target antigen-binding domain e.g., huUCHTl
  • a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain wherein the order is 5’ end to 3’ end.
  • a nucleic acid disclosed herein is in an order of (1) a target antigen-binding domain; (2) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 3’ end to 5’ end.
  • a target antigen-binding domain e.g., huUCHTl
  • a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain wherein the order is 3’ end to 5’ end.
  • a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain.
  • a UCHT1 e.g., huUCHTl
  • a target antigen-binding domain e.g., huUCHTl
  • a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 5’ end to 3’ end.
  • a UCHT1 e.g., huUCHTl
  • a target antigen-binding domain e.g., a target antigen-binding domain
  • a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain e.g., CD4 transmembrane domain and a cytosolic domain
  • a nucleic acid described herein is in an order of (1) a polynucleotide encoding a UCHT1 (e.g., huUCHTl); (2) a target antigen-binding domain; (3) a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain, wherein the order is 3’ end to 5’ end.
  • a UCHT1 e.g., huUCHTl
  • a target antigen-binding domain e.g., a target antigen-binding domain
  • a polynucleotide encoding a CD4 transmembrane domain and a cytosolic domain e.g., CD4 transmembrane domain and a cytosolic domain
  • the target antigen-binding domain, the UCHT1 (e.g., huUCHTl), and CD4 transmembrane domain and a cytosolic domain polypeptides are directly fused.
  • the target antigen-binding domain and the CD4 transmembrane domain and a cytosolic domain polypeptide are both fused to the UCHT1 (e.g, huUCHTl).
  • the target antigen-binding domain, the UCHT1 (e.g., huUCHTl), and CD4 transmembrane domain and a cytosolic domain polypeptides are joined by at least one linker.
  • the first polypeptide and the second polypeptide are directly fused, and joined to the third polypeptide by a linker. In some embodiments, the second polypeptide and the third polypeptide are directly fused, and joined to the first polypeptide by a linker.
  • the linker is a peptide linker. In some embodiments, the peptide linker comprises 1 to 40 amino acids. In some embodiments, the peptide linker comprises 1 to 30 amino acids. In some embodiments, the peptide linker comprises 1 to 15 amino acids. In some embodiments, the peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 1 to 6 amino acids.
  • the peptide linker comprises 30 to 40 amino acids. In some embodiments, the peptide linker comprises 32 to 36 amino acids. In some embodiments, the peptide linker comprises 5 to 30 amino acids. In some embodiments, the peptide linker comprises 5 amino acids. In some embodiments, the peptide linker comprises 10 ammo acids. In some embodiments, the peptide linker comprises 15 ammo acids. In some embodiments, the peptide linker comprises 20 amino acids. In some embodiments, the peptide linker comprises 25 amino acids. In some embodiments, the peptide linker comprises 30 amino acids.
  • the at least one linker comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 41 (flexible connector), SEQ ID NO: 45 (G4S flexible linker), or SEQ ID NO: 46 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 22 (short helix connector), SEQ ID NO: 24 (long helix connector), SEQ ID NO: 26 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 96% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 97% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the at least one linker comprises the amino acid sequence of SEQ ID NO: 18 ((G4S)4-based linker), SEQ ID NO: 20 (G4S-based linker), SEQ ID NO: 26 (CD4 based linker), SEQ ID NO: 28 (short helix connector), SEQ ID NO: 30 (long helix connector), SEQ ID NO: 32 (large domain connector), SEQ ID NO: 209 (flexible connector), SEQ ID NO: 211 (G4S flexible linker), or SEQ ID NO: 213 (G4S3 flexible linker).
  • the peptide linker that joins the target antigen-binding domain to the antigen-binding domain that binds a TCR complex is known as the connector to distinguish this protein domain from other linkers in the TAC.
  • the connector may be of any size.
  • the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a short helix comprising SEQ ID NO: 28.
  • the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a short helix encoded by SEQ ID NO: 27.
  • the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a long helix comprising SEQ ID NO: 29. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a long helix encoded by SEQ ID NO: 28. In some embodiments, the connector between the antigen-binding domain that binds a TCR complex and the target antigen-binding domain is a large domain comprising SEQ ID NO: 32.
  • a nucleic acid or TAC disclosed herein comprises a leader sequence.
  • the leader sequence is encoded by a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCDSa leader).
  • the leader sequence is encoded by a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).
  • the leader sequence is encoded by a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).
  • the leader sequence is encoded by a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).
  • the leader sequence is encoded by a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 33 (mulgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).
  • the leader sequence comprises the nucleotide sequence of SEQ ID NO: 33 (muIgG leader), SEQ ID NO: 35 (huIgG leader), or SEQ ID NO: 37 (huCD8a leader).
  • a nucleic acid or TAC disclosed herein comprises a leader sequence.
  • the leader sequence comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).
  • the leader sequence comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).
  • the leader sequence comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).
  • the leader sequence comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).
  • the leader sequence comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader). In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 34 (muIgG leader), SEQ ID NO: 36 (huIgG leader), or SEQ ID NO: 38 (huCD8a leader).
  • the T cell receptor signaling domain polypeptide comprises a TCR co-receptor domain.
  • the TCR signaling domain polypeptide comprises a transmembrane domain and/or a cytosolic domain of a TCR co-receptor.
  • the TCR co-receptor is CD4, CD8, LAG3, or a chimeric variation thereof.
  • the TCR co-receptor is CD4.
  • the TAC comprises a transmembrane domain and a cytosolic domain of a CD4 co-receptor.
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 96% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 97% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 98% sequence identity with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity' with the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 41 (CD4 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 85% sequence identity with the ammo acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 42 (CD4 transmembrane and cytosolic domain).
  • the TCR co-receptor is CD8. In some embodiments, the TCR coreceptor is CD8a. In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain. In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 43 (CD8 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CDS transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 44 (CD8 transmembrane and cytosolic domain).
  • the TCR signaling domain polypeptide comprises a chimera of sequences or domains from co-receptors.
  • the TCR signaling domain polypeptide comprises a chimera of CD8a and CD8P, wherein the CD8a arginine rich region is replaced with the CD8P arginine rich region (CD8a+R(P) chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain composes a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 45 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 46 (CD8a+R(P) chimera).
  • the TCR signaling domain polypeptide comprises a chimera of CD8a and CD8P, where the CD8a CXCP domain, which contains an Lek binding motif, is appended to the C-terminus of the CD8P cytosolic domain (CD8P+Lck chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8P+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the polynucleotide encoding the cytosolic and transmembrane domain comprises a nucleotide sequence having at least 99% sequence identity with the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera).
  • the polynucleotide encoding the cytosolic and transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 47 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera).
  • the cytosolic and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 48 (CD8p+Lck chimera). In some embodiments, the cytosolic and transmembrane domain comprise the amino acid sequence of SEQ ID NO: 48 (CD8P+Lck chimera).
  • the TCR signaling domain polypeptide includes both a cytosolic domain and a transmembrane domain of a TCR co-receptor protein.
  • the cytosolic domain and transmembrane domain are from the same co-receptor or from different co-receptors.
  • the target antigen-binding domain refers to any substance or molecule that binds, directly or indirectly, to a target antigen.
  • Target-specific antigen-binding domains include, but are not limited to, antibodies and fragments thereof, for example single chain antibodies such as single-chain antibodies (scFvs), single domain antibodies (e.g., nanobodies), peptides, peptidomimetics, proteins, glycoproteins, or proteoglycans that bind to the target cell and/or antigen.
  • the target-specific ligands include, but are not limited to, designed ankyrin repeat proteins (DARPins), lectins, knottins, centryrins, anticalins, or naturally occurring ligands for the tumor antigen, such as growth factors, enzyme substrates, receptors or binding proteins.
  • target specific ligands include non-protein compounds that bind to target cells and/or antigens, including but not limited to carbohydrates, lipids, nucleic acids, or small molecules.
  • a target-specific ligand is a designed ankyrin repeat protein (DARPin) targeted to a specific cell and/or antigen.
  • DARPin designed ankyrin repeat protein
  • a target-specific ligand is a single-chain variable fragment (scFv) targeted to a specific cell and/or antigen, antigen.
  • a target-specific ligand is a nanobody (VHH) targeted to a specific cell and/or antigen.
  • the target antigen is expressed on a cancer/tumor cell. In some embodiments, the target antigen is expressed on a metastatic cancer cell. In some embodiments, the target antigen is human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), cluster of differentiation 19 (CD19), Claudin-18.2 (CLDN18.2), guanylate cyclase 2C (GUCY2C), or glypican 3 (GPC3).
  • HER2 human epidermal growth factor receptor 2
  • BCMA B cell maturation antigen
  • CD19 Claudin-18.2
  • Claudin-18.2 Claudin-18.2
  • GUI2C guanylate cyclase 2C
  • GPC3 glypican 3
  • the target antigen is HER2.
  • the HER2- binding domain comprises an antigen-binding domain of an antibody selected from Trastuzumab, Pertuzumab, Lapatinib, Neratinib, Ado-trastuzmab Emtansine, Gancotamab, Margetuximab, Timigutuzumab, and Ertumaxomab.
  • the HER2-binding domain is a DARPin that specifically binds a HER2 antigen.
  • the DARPin targeted to HER2 comprises SEQ ID NO: 49.
  • the HER2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2- bmding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2 -binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence having at least 95% sequence identity' with SEQ ID NO: 49. In some embodiments, the HER2-binding domain comprises an amino acid sequence of SEQ ID NO: 49.
  • the target antigen is BCMA.
  • the BCMA- binding domain comprises an antigen-binding domain of an antibody selected from Belantamab mafodotin, and GSK2857916.
  • the target-binding domain is an scFv that specifically binds BCMA.
  • the scFv that binds BCMA comprises SEQ ID NO: 50.
  • the BCMA-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 50. In some embodiments, the BCMA-binding domain comprises an amino acid sequence of SEQ ID NO: 50.
  • the target antigen is CD19.
  • the CD19- binding domain is an scFv that selectively binds a CD 19 antigen.
  • the scFv targeted to CD 19 comprises SEQ ID NO: 51.
  • the CD19-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 51.
  • the CD19- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 51.
  • the CD19-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51.
  • the CD19-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 51. In some embodiments, the CD19-bindmg domain comprises an ammo acid sequence having at least 95% sequence identity' with SEQ ID NO: 51. In some embodiments, the CD19-binding domain comprises an amino acid sequence of SEQ ID NO: 51.
  • the target antigen is Claudin 18.2.
  • the Claudin 18.2-binding domain is a nanobody that selectively binds a Claudin 18.2 antigen.
  • the scFv targeted to Claudin 18.2 comprises SEQ ID NO: 52 or 53.
  • the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 52. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 52.
  • the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an ammo acid sequence having at least 80% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 53. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 53.
  • the target antigen is GUCY2C.
  • the GUCY2C -binding domain is an scFv that selectively binds a GUCY2C antigen.
  • the scFv targeted to GUCY2C comprises any one of SEQ ID NOs: 54-117.
  • the GUCY2C -binding domain is a nanobody that selectively binds a GUCY2C antigen.
  • the scFv targeted to GUCY2C comprises any one of SEQ ID NOs: 118-136.
  • the GUCY2C-binding domain comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C -binding domain comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 54-117.
  • the GUCY2C -binding domain comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 54- 117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 54-117. In some embodiments, the GUCY2C-binding domain comprises an amino acid sequence of SEQ ID NO: 54-117.
  • the Claudin 18.2-binding domain comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an ammo acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 118-136.
  • the Claudin 18.2-binding domain comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 118-136. In some embodiments, the Claudin 18.2-binding domain comprises an amino acid sequence of SEQ ID NO: 118-136.
  • the target antigen is GPC3.
  • the GPC3- binding domain is a nanobody that selectively binds a GPC3 antigen.
  • the scFv targeted to GPC3 comprises SEQ ID NO: 137.
  • the GPC3-binding domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3- binding domain comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence having at least 95% sequence identity' with SEQ ID NO: 137. In some embodiments, the GPC3-binding domain comprises an amino acid sequence of SEQ ID NO: 137.
  • the TAC disclosed herein is a HER2-TAC.
  • the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 138. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 138.
  • the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC compnses an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 139. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 139.
  • the HER2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 140. In some embodiments, the HER2-TAC comprises an amino acid sequence of SEQ ID NO: 140.
  • the TAC disclosed herein is a BCMA-TAC.
  • the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity' with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 141. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 141.
  • the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an ammo acid sequence having at least 80% sequence identity' with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 142. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 142.
  • the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity' with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 143. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 143.
  • the BCMA-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 80% sequence identity' with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 144. In some embodiments, the BCMA-TAC comprises an amino acid sequence of SEQ ID NO: 144.
  • the TAC disclosed herein is a CD19-TAC.
  • the CD19-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 145.
  • the CD19-TAC comprises an ammo acid sequence having at least 75% sequence identity with SEQ ID NO: 145.
  • the CD19- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 145.
  • the CD19-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 145.
  • the CD19-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 145.
  • the CD19-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 145.
  • the CD19-TAC comprises an amino acid sequence of SEQ ID NO: 145.
  • the TAC disclosed herein is a Claudin 18.2-TAC.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity' with SEQ ID NO: 146.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 146.
  • the Claudin 18.2-TAC comprises an ammo acid sequence having at least 80% sequence identity with SEQ ID NO: 146.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 146.
  • the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity' with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 146. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 146.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 147. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 147.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an ammo acid sequence having at least 85% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 148. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 148.
  • the Claudin 18.2-TAC comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2- TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 149. In some embodiments, the Claudin 18.2-TAC comprises an amino acid sequence of SEQ ID NO: 149.
  • the TAC disclosed herein is a GUCY2C-TAC.
  • the GUCY2C-TAC comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 150-206.
  • the GUCY2C- TAC comprises an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs: 150-206.
  • the GUCY2C-TAC comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 150-206.
  • the GUCY2C-TAC comprises an amino acid sequence having at least 85% sequence identity' with any one of SEQ ID NOs: 150-206.
  • the GUCY2C- TAC an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 150-206. In some embodiments, the GUCY2C-TAC comprises an amino acid sequence of any one of SEQ ID NOs: 150-206.
  • the TAC disclosed herein is a GPC3-TAC.
  • the GPC3-TAC comprises an amino acid sequence having at least 70% sequence identity' with SEQ ID NO: 207.
  • the GPC3-TAC comprises an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 207.
  • the GPC3- TAC comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 207.
  • the GPC3-TAC comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 207.
  • the GPC3-TAC an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 207.
  • the GPC3-TAC comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 207.
  • the GPC3-TAC comprises an amino acid sequence of SEQ ID NO: 207.
  • the TACs disclosed herein are expressed in T cells (e.g., a(3 T cells, y5 T cells).
  • TAC-expressing T cells disclosed herein are administered to subjects in need thereof as part of a combination therapy.
  • the combination therapy comprises co-administration of an immune-checkpoint inhibitor.
  • Immune checkpoint inhibitors can be used to support sustained activation of engineered T cells, for example, by blocking negative feedback loops that suppress effector T cell activation.
  • PD-1 immune checkpoint One example of a negative feedback loop suppressing sustained T cell activation is the PD-1 immune checkpoint.
  • T cells increase expression of programmed death protein 1 (PD-1 or PD1), which can bind to its ligands PD-1 ligand 1 (PD-L1) or PD-1 ligand 2 (PD-L2) expressed on normal cells.
  • PD-1 or PD1 programmed death protein 1
  • PD-L1 PD-1 ligand 1
  • PD-L2 PD-1 ligand 2
  • cancer cells often upregulate PD-L1 expression as a mechanism of immune evasion inhibiting and ultimately causing the accumulation of functionally exhausted T cells.
  • immune checkpoints can therefore be used to protect T cells from inhibition and exhaustion induced by factors such as PD-1/PD-L1 binding.
  • Other immune checkpoint proteins include CTLA-4, LAG3, BTLA, B7- H3, B7-H4, TIM-3, and KIR.
  • the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a BTLA inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, or a KIR inhibitor.
  • the immune checkpoint inhibitor is a PD-1 inhibitor.
  • the immune checkpoint inhibitor is a PD-L1 inhibitor.
  • the immune checkpoint inhibitor is a PD- L2 inhibitor.
  • the immune checkpoint inhibitor is a CTLA-4 inhibitor.
  • the immune checkpoint inhibitor is a LAG3 inhibitor.
  • the immune checkpoint inhibitor is a TIM-3 inhibitor. In some embodiments, the immune checkpoint inhibitor is a BTLA inhibitor. In some embodiments, the immune checkpoint inhibitor is aB7-H3 inhibitor. In some embodiments, the immune checkpoint inhibitor is a B7-H4 inhibitor. In some embodiments, the immune checkpoint inhibitor is a KRI inhibitor.
  • the immune checkpoint inhibitor is an antibody or antigenbinding fragment derived therefrom. In some embodiments, the immune checkpoint inhibitor is a small molecule.
  • the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, or relatlimab.
  • the immune checkpoint inhibitor is nivolumab.
  • the immune checkpoint inhibitor is pembrolizumab.
  • the immune checkpoint inhibitor is cemiplimab.
  • the immune checkpoint inhibitor is atezolizumab.
  • the immune checkpoint inhibitor is avelumab.
  • the immune checkpoint inhibitor is durvalumab.
  • the immune checkpoint inhibitor is ipilimumab.
  • the immune checkpoint inhibitor is tremelimumab.
  • the immune checkpoint inhibitor is relatlimab.
  • the TACs disclosed herein are delivered to a T cell via a vector.
  • the vectors further comprise a promoter.
  • the promoter is functional in a mammalian cell. Promoters, regions of DNA that initiate transcription of a particular nucleic acid sequence, are well known in the art.
  • a “promoter functional in a mammalian cell” refers to a promoter that drives expression of the associated nucleic acid sequence in a mammalian cell.
  • a promoter that drives expression of a nucleic acid sequence is referred to as being “operably connected” to the nucleic acid sequence.
  • a variety of delivery vectors and expression vehicles are employed to introduce nucleic acids described herein into a cell.
  • the TAC coding sequence is operably connected to the promoter.
  • the vector is designed for expression in mammalian cells such as T cells.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector.
  • vectors that are useful comprise vectors derived from lentiviruses, Murine Stem Cell Viruses (MSCV), pox viruses, oncoretroviruses, adenoviruses, and adeno- associated viruses.
  • Other delivery vectors that are useful comprise vectors derived from herpes simplex viruses, transposons, vaccinia viruses, human papilloma virus, Simian immunodeficiency viruses, HTLV, human foamy virus and variants thereof.
  • vectors that are useful comprise vectors derived from spumaviruses, mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, mammalian type D retroviruses and HTLV/BLV type retroviruses.
  • a lentiviral vector useful in the disclosed compositions and methods is the pCCL4 vector.
  • the T cell is transduced or transfected with a nucleic acid sequence encoding a HER2 T cell antigen coupler, for example a vector comprising a nucleic acid sequence encoding a HER2.
  • the T cell is an isolated T cell.
  • T cells are obtained from a number of sources, including, but not limited to blood (for example, peripheral blood mononuclear cells), bone marrow, thymus tissue, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, spleen tissue, or tumors.
  • blood for example, peripheral blood mononuclear cells
  • bone marrow for example, thymus tissue, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, spleen tissue, or tumors.
  • the T cells are autologous T cells. In some embodiments, the T cells are allogenic. In some embodiments, the T cells are a(3 (alpha beta) T cells (z.e., T cell with a TCR composed of one a (alpha) chain and (beta) chain. In some embodiments, the T cells are gamma delta (y5) T cells (i.e. , T cells with a TCR composed of one y (gamma) chain and one 5 (delta) chain.
  • the T cells are obtained from a cell line of T cells.
  • the T cells are obtained from donors (allogeneic T cells).
  • the T cells are obtained by differentiation of embryonic or adult stem cells or from induced pluripotent stem cells.
  • the T cells regardless of the source of T cells, the T cells have been modified so that they lack expression of an endogenous TCR and/or permanently or transiently lack expression of MHC/HLA molecules (universal donor T cells).
  • the T cells are autologous with respect to the subject.
  • the cells are allogeneic, syngeneic, or xenogeneic with respect to the subject.
  • the T cells are optionally enriched in vitro.
  • a population of cells is enriched by positive or negative selection.
  • the T cells are optionally frozen or cryopreserved and then thawed at a later date.
  • T cells are activated and/or expanded before or after introducing the TAC to the T cells.
  • the T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulator molecule on the surface of the T cells.
  • the T cells are expanded by contact with one or more soluble agents that stimulate CD3/TCR complex signaling and co-stimulator molecule signaling.
  • the T cells are transduced or transfected with nucleic acid sequences.
  • the transduced or transfected T cells express proteins coded for by the transfected or transduced nucleic acid sequences.
  • a nucleic acid may be introduced into a cell by physical, chemical, or biological means. Physical means include, but are not limited to, microinjection, electroporation, particle bombardment, lipofection and calcium phosphate precipitation.
  • Bio means include the use of DNA and RNA vectors.
  • Viral vectors are used to introduce and express a nucleic acid into a T cell.
  • Viral vectors include vectors derived from lentivirus, Murine Stem Cell Viruses (MSCV), pox viruses, herpes simplex virus I, adenovirus and adeno-associated viruses.
  • the vector optionally includes a promoter that drives expression of the transduced nucleic acid molecule in a T cell (e.g, a CMV promoter, eFla promoter, or MSCV promoter).
  • any suitable assay is used to confirm the presence and/or expression of the transduced nucleic acid sequence and/or the polypeptide encoded by the nucleic acid in the T cell.
  • Assays include, but are not limited to, Southern and Northern blotting, RT-PCR and PCR, ELISA, Western blotting, and flow cytometry.
  • a T cell expressing a TAC has increased T cell activation in the presence of an antigen compared to a T cell not expressing a TAC and/or as compared to a T cell expressing a traditional CAR.
  • Increased T cell activation is ascertained by numerous methods, including but not limited to, increased tumor cell line killing, increased cytokine production, increased cytolysis, increased degranulation and/or increased expression of activation markers such as CD107a, IFNy, IL2 or TNFa.
  • increases are measured in an individual cell or in a population of cells.
  • the terms “increased” or “increasing” as used herein refer to at least a 1%, 2%, 5%, 10%, 25%, 50%, 100% or 200% increase in a T cell or population of T cells expressing a TAC compared to a T cell or population of T cells not expressing a TAC and/or as compared to a T cell or population of T cells expressing a traditional CAR.
  • the T cell expressing the HER2 TAC is administered to an individual in need in a pharmaceutical compositions comprising a HER2 TAC T cell (e.g., an a[l T cell, a y5 T cell) disclosed herein (transduced with and/or expressing a TAC), and a pharmaceutically acceptable carrier.
  • a HER2 TAC T cell e.g., an a[l T cell, a y5 T cell
  • a pharmaceutically acceptable carrier e.g., a[l T cell, a y5 T cell
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants ⁇ e.g., aluminum hydroxide); or preservatives.
  • the engineered T cells e.g., a
  • compositions are administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration is determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages are determined by clinical trials.
  • an immunologically effective amount “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated
  • the precise amount of the compositions of the present invention to be administered is determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • the modified T cells e.g., 0.(3 T cells, y6 T cells
  • the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of 10 1 to 10 1 ’ cells/kg body weight, 10 4 to 10 9 cells/kg body weight, optionally 10 5 to 10 8 cells cells/kg body weight, 10 6 to IO 7 cells/kg body weight or 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of greater than 10 1 cells/kg body weight.
  • the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage of less than 10 15 cells/kg body weight.
  • the modified T cells are administered at a dosage of about 6* 10 4 to about 6* 10 8 cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about I x lO 5 to about 8xio 6 cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6xl0 4 to 8xl0 4 cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about I x lO 5 to 3*10 5 cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6x l0 5 to 8x l0 5 cells/kg body weight.
  • the modified T cells are administered at a dosage of about 1 xio 6 to 3xl0 6 cells/kg body weight. In some embodiments, the modified T cells are administered at a dosage of about 6xl0 6 to 8x l0 6 cells/kg body weight.
  • the modified T cells e.g., a0 T cells, y5 T cells
  • pharmaceutical compositions described herein are administered at a dosage of 0.5 x 10 4 cells, I x lO 4 cells, 2xl0 4 cells, 4 l0 4 cells, 0.5x l0 5 cells, 2xl0 5 cells, 4xl0 5 cells, 0.5xl0 6 cells, 2xl0 6 cells, 4xl0 5 cells, 5xl0 6 cells, 1.2xl0 7 cells, 2xl0 7 cells, 5xl0 7 cells, 2x l0 8 cells, 5xl0 8 cells, or 2x l0 9 cells.
  • the modified T cells and/or pharmaceutical compositions described herein are administered at a dosage in a range of 0.005-2000x 10 6 cells, 0.01-2000x 10 6 cells, 0.025-2000xl0 6 cells, 0.05-2000xl0 6 cells, 0.1-2000x l0 6 cells, 0.25-2000xl0 6 cells, 0.5- 2000xl0 6 cells, 0.5-2xl0 4 cells, 0.5-2xl0 5 cells, 0.5-2xl0 6 cells, 0.5-2xl0 7 cells, 0.5-2x l0 8 cells, or 0.5-2xl0 9 cells, including all integer values within those ranges.
  • compositions comprising engineered/modified and unmodified T cells (e.g., ot(3 T cells, y6 T cells), or comprising different populations of engineered/modified T cells with or without unmodified T cells.
  • engineered/modified and unmodified T cells e.g., ot(3 T cells, y6 T cells
  • a therapeutic quantity of engineered/modified T cells need not be homogenous in nature.
  • activated engineered/modified T cells further activate unmodified T cells (e.g., a bystander T cell) within the same pharmaceutical composition/cell population (referred hereinto as the “bystander effect”).
  • engineered/modified T cells activate unmodified T cells only when activated in response to binding of the antigen to the TAC expressed by the engineered/modified T cell.
  • T cell e.g., an a.f> T cell, a yd T cell
  • compositions are administered multiple times at these dosages.
  • the dosage is administered a single time or multiple times, for example daily, weekly, biweekly, or monthly, hourly, or is administered upon recurrence, relapse or progression of the cancer being treated.
  • the cells in some embodiments, are administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the modified T cells e.g., a
  • an immune checkpoint inhibitor e.g., nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, and durvalumab.
  • the immune checkpoint inhibitor is pembrolizumab.
  • pembrolizumab is administered at a dose of about 0.1-1 mg/kg, 1- 5 kg/mg, 1-10 kg/mg, 1-15 kg/mg, 5-10 kg/mg, 5-15 kg/mg, or 10-15 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 1 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 2 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 3 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 4 kg/mg.
  • pembrolizumab is administered at a dose of about 5 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 6 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 7 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 8 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 9 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 10 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 11 kg/mg.
  • pembrolizumab is administered at a dose of about 12 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 13 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 14 kg/mg. In some embodiments, pembrolizumab is administered at a dose of about 15 kg/mg.
  • pembrolizumab is administered as a single dose. In some embodiments, the pembrolizumab is administered over multiple doses. In some embodiments, multiple equivalent dose amounts of pembrolizumab are administered. In some embodiments, multiple different dose amounts of pembrolizumab are administered.
  • the dosage interval is about 1-2 days, 2-3 days, 3-5 days, 4-5 days, 5-6 days, 6-7 days, 1-2 weeks, 2-3 weeks, 3-4 weeks, or 4-5 weeks for subsequent doses to the first dose. In some embodiments, the dosage interval is approximately 4-5 days for subsequent doses to the first dose.
  • the first dose is administered at a dose of about 1-10 kg/mg. In some embodiments, the first dose is administered at a dose of about 10 kg/mg. In some embodiments, the first dose is administered at a dose of about 9 kg/mg. In some embodiments, the first dose is administered at a dose of about 8 kg/mg. In some embodiments, the first dose is administered at a dose of about 7 kg/mg. In some embodiments, the first dose is administered at a dose of about 6 kg/mg. In some embodiments, the first dose is administered at a dose of about 5 kg/mg. In some embodiments, the first dose is administered at a dose of about 4 kg/mg.
  • the first dose is administered at a dose of about 3 kg/mg. In some embodiments, the first dose is administered at a dose of about 2 kg/mg. In some embodiments, the first dose is administered at a dose of about 1 kg/mg.
  • the subsequent dose is administered at a dose of about 1-10 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 10 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 9 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 8 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 7 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 6 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 5 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 4 kg/mg.
  • the subsequent dose is administered at a dose of about 3 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 2 kg/mg. In some embodiments, the subsequent dose is administered at a dose of about 1 kg/mg. In some embodiments, a first 10 mg/kg dose of pembrolizumab is followed by subsequent dose of 5 mg/kg of pembrolizumab every 4-5 days over a full course of treatment.
  • the pharmaceutical composition is substantially free of, e.g, there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti- CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium a fungus, mycoplasma, IL-2, and IL-7.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti- CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium
  • engineered T-cells disclose herein are administered to a subject and blood is subsequently redrawn (or apheresis performed), T-cells therefrom are activated and reinfused into the patient with engineered T cells. This process, in some embodiments, is carried out multiple times every few weeks. T-cells are activated from blood draws of from 10 cc to 400 cc. T-cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
  • the modified/engineered T cells and/or pharmaceutical compositions are administered by methods including, but not limited to, aerosol inhalation, injection, infusion, ingestion, transfusion, implantation or transplantation.
  • the modified T cells and/or pharmaceutical compositions are administered to a subject transarterially, subcutaneously, intradermally, intratumorally, intranodally, intrameduliary, intramuscularly, by intravenous (i.v.) injection, by intravenous (i.v.) infusion, or intraperitoneally.
  • the modified/engineered T cells and/or pharmaceutical compositions thereof are administered to a patient by intradermal or subcutaneous injection.
  • the modified/engineered T cells and/or pharmaceutical compositions thereof are administered by i.v. injection.
  • the modified/engineered T cells and/or pharmaceutical compositions thereof are injected directly into a tumor, lymph node, or site of infection.
  • the modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.
  • the modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of at greater than at most about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.
  • the modified/engineered T cells T cells and/or pharmaceutical compositions are administered in a volume of at least about 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 200 mL, 300 mL, 400 mL, or 500 mL.
  • a pharmaceutical composition is prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that are administered to subjects, such that an effective quantity of the T cells is combined in a mixture with a pharmaceutically acceptable carrier.
  • Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., USA, 2000).
  • the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable carriers or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
  • suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, N-(l(2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes.
  • DOTMA N-(l(2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride
  • DOPE diolesylphosphotidyl-ethanolamine
  • liposomes include a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.
  • compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
  • Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
  • a pharmaceutical composition disclosed herein is formulated into a variety of forms and administered by a number of different means.
  • a pharmaceutical formulation is administered orally, rectally, or parenterally, in formulations containing conventionally acceptable carriers, adjuvants, and vehicles as desired.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, or intrastemal injection and infusion techniques.
  • Administration includes injection or infusion, including intra-arterial, intracardiac, intracerebroventricular, intradermal, intraduodenal, intramedullary , intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalational, transdermal, transmucosal, sublingual, buccal and topical (including epicutaneous, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration.
  • a route of administration is via an injection such as an intramuscular, intravenous, subcutaneous, or intraperitoneal injection.
  • Liquid formulations include an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, an aerosol, and the like. In certain embodiments, a combination of various formulations is administered. In certain embodiments a composition is formulated for an extended-release profile.
  • T cells were engineered to express aHER2-TAC (SEQ ID NO: 36) and injected at a dosage of 6xl0 6 TAC+ T cells into NSG mice bearing HER2-positive NCI-N87 subcutaneous xenografts once tumors reached a size of approximately 100 mm 3 .
  • Non-transduced T cells were used as negative control.
  • 30 minutes following T cell engraftment half of mice in treatment and control groups were sacrificed and tissues and blood samples were collected, at which point TAG T cells were not detected in the tumor. Tumor volumes were monitored in remaining mice for 7 days, after which, mice were sacrificed, and tissues were collected and further processed to isolate HER2-TAC T cells for subsequent flow cytometry analysis.
  • FIG. 1A A summary of the experiment is depicted in FIG. 1A.
  • tumor spheroids were created with HER2 + , GFP-expressing N87 cancer cells grown in the presence of human dermal fibroblasts.
  • Spheroids were treated with T cells engineered to express a HER2-TAC (SEQ ID NO: 36) at effector: target (E:T) ratios of 2: 1, 10: 1, and 20: 1 and observed over 4 days.
  • Non-transduced T cells were used at the same ratios as negative controls.
  • Spheroids were also cultured in media only (negative control) or 0.5 pM Staurosporin (STS) (positive control). Spheroids were imaged daily by fluorescence microscopy.
  • spheroids treated with TAC T cells showed a marked reduction in size, particularly at E:T ratios of 10: 1 and 20: 1 (FIG. 2A), whereas there was no discernable reduction of spheroid size in NTD control groups. Furthermore, spheroid cells treated with TAC T cells had reduced percent viability at all E:T ratios based on reduction of GFP fluorescence, as did SPS-treated spheroids. By contrast, NTD control groups did not show any reduced viability (FIG. 2B). Spheroids treated with control or TAC-expressing T cells at an E:T ratio of 2: 1 were fixed with 4% paraformaldehyde after four days of treatment and analyzed via immunohistochemistry.
  • HER2 expression was confirmed in all tissues.
  • T cell infiltration (CD3) was only observed in HER2-TAC T treated cells.
  • TAC T cell infiltration was accompanied with a significant upregulation of PD-L1 (PD-L1) as well as the secretion of Granzyme B and activation of Caspase 3 (cleaved caspase 3) (FIG. 2C).
  • HER2-TAC T cells are capable of infiltrating spheroid tissues and indicates T cell induced tumor cell death via the caspase pathway. Concomitantly, a significant upregulation of PD-L1 in tumor tissue was observed. Since PD-L1 can interact with PD-1 expressed on activated T cells and inhibit T cell function, blocking PD-1 may counteract PD-1 -induced T cell inhibition and ultimately exhaustion and, consequently, increase the potency of TAC T cells.
  • Example 2 In vitro analysis of an anti-PD-1 antibody/TAC T cell combination therapy [0164] HER2-expressing cancer cell lines BT-474 (breast cancer) and N87 (gastric carcinoma) were engineered to overexpress PD-L1. This led to a significant upregulation in PD-L1 in both BT-474 (BT-474 PD ‘ L1/H ' gh ) (FIG. 3A) and N87 (N87 PD - L1/Higl1 ) (FIG. 3B) cells relative to the nonengineered parental cell lines.
  • BT-474 breast cancer
  • N87 gastric carcinoma
  • HER2-TAC T cells or non-transduced control T cells were incubated with Cell Trace Violet (CTV) and co-cultured with BT-474, and BT- anc
  • CTV Cell Trace Violet
  • HER2-expressing N87 WT/nuc GFP or N87 PD ' L14Ilgh/nuc GFP cancer cells were co-cultured with HER2-TAC T cells, administered with or without anti-PD-1 antibody pembrolizumab, and assessed for viability (FIG. 4A-4B).
  • target N87 WT tumor cells co-cultured with HER2-TAC T cells dense cell clusters were observed and, in contrast to the NTD controls, large areas of the cell culture well were devoid of tumor cells, demonstrating target cell killing in both (“Untreated”) or treated with pembrolizumab (“+Pembro”).
  • target N87 PD ' L1 ' Hlgh tumor cells co-cultured with HER2-TAC T cells little clearing was seen in the untreated well (“Untreated”), while in comparison, the pembrolizumab-treated well (“+Pembro”) displayed significantly cell clustering and large areas where no tumor cells could be detected, demonstrating extensive N87 PD ' L1 ' Hlgh tumor cell death in the pembrolizumab-treated well.
  • FIG. 4A The cell imaging results (FIG. 4A) and quantification of percentage of live cancer cells (FIG. 4B) demonstrate that HER2-TAC T cell treatment of PD-L1 expressing tumor cells, administered in combination with pembrolizumab, had a positive impact on reducing the number of live PD-L1 high tumor cells over the course of the experiment, thereby indicating that PD1 signaling blockade can increase the potency of TAC T cells in the presence of tumor cells with high PD-L1 levels.
  • TAC T cells are produced through genetic engineering, incorporating TAC receptors into a patient’s own T cells. This redirects these enhanced T cells to target specific cancer antigens; and upon tumor recognition, activate them by co-opting the natural signaling pathways of the T cell receptor (TCR).
  • TCR T cell receptor
  • TAC T cells use HER2 antigens, present on the surface of cancer cells, to recognize and eradicate tumor cells. This approach has resulted in promising outcomes in mouse models, demonstrating TAC T cells accumulate within solid tumors and lead to robust anti-tumor efficacy, while yielding a favorable safety profile. Importantly, TAC T cells can persist for extended periods of time in mice and protect the host from tumor regrowth. Consequently, it is hypothesized TAC T cell monotherapy will be safe and effective in treating patients with HER2 + solid tumors and can provide a significant therapeutic benefit in an area of high unmet medical need.
  • the dose finding portion of this trial (Phase 1) will evaluate increasing dose levels of TAC T cells used as a monotherapy, and in combination with pembrolizumab, to identify recommended Phase 2 doses (RP2Ds) in patients with solid tumors who are HER2+ (with immunohistochemistry [IHC] expression levels of 3+, 2+, and 1+) and have progressed after 2 lines of systemic therapy.
  • the combination arm will specifically investigate the safety and clinical activity of adding an anti-PDl checkpoint inhibitor to TAC T cell monotherapy.
  • pembrolizumab may enhance TAC T cell activity and antitumor potency, while preventing TAC T cell exhaustion, through attenuation of inhibitory pathways which occur once PD-1 receptors on T cells bind to PD-L1 and PD-L2 ligands expressed on cancer cells and other immune cells.
  • Phase 2 the following dose expansion groups will further evaluate the safety', efficacy, and pharmacokinetics (PK) of TAC T cell monotherapy and TAC T cells + pembrolizumab combination therapy at the RP2Ds: breast, lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others.
  • PK pharmacokinetics
  • HER2 amplification is known to occur in breast, gastric, salivary, vaginal, endometrial, bladder, colorectal, and cervical cancers, activating numerous oncogenic signaling axes (e.g. , PI3K/AKT and Ras/Raf/ERK) resulting in improved malignant cell survival, proliferation, migration, and resistance to immunotherapy. Novel therapies are urgent needed to more effective treat these patient populations.
  • oncogenic signaling axes e.g. , PI3K/AKT and Ras/Raf/ERK
  • PD-1 Programmed cell death protein 1
  • pembrolizumab treatment i.e., anti-PD-1 monoclonal antibody
  • Tumors that are associated with improved clinical responses to checkpoint blockade are those with known high PD-L1 expression, however responses can also be observed in tumors without PD-L1 expression (Garon et al. N Engl J Med. 2015; 372(21) and Topalian et al. N Engl J Med. 2012; 366(26)).
  • PD-L1 expression is believed to be induced in these tumor cells, following the influx of T cell infiltrate and after reinvigoration via checkpoint blockade, through the secretion of T cell cytokines, IFN-y and TNF-a (Spranger et al. Sci Transl Med. 2013;
  • TAC T cell activity was reduced when ectopic PD-L1 expression was high relative to a parental line expressing low PD-L1 levels.
  • upregulation of PD-1 receptors has been observed on TAC T cells following antigen-specific activation.
  • pembrolizumab has recently been demonstrated with trastuzumab in metastatic oesophagogastric cancer (O’Donnell et al. Nat Rev Clin Oncol. 2019; 16(3)), and margetuximab in gastro-oesophageal adenocarcinoma (Catenacci et al. Lancet Oncol. 2020; 21(8)).
  • Pembrolizumab has also been safety added to CD19-directed CAR T cell therapy in B cell lymphomas (Chong et al. Blood.
  • TAC T cells can also be used in combination with pembrolizumab to yield safe and more effective outcomes in patients with solid tumors.
  • TAC T cells 27 total patients were infused with CAR T cells: 25 had malignant pleural mesothelioma (MPM), and 1 each had metastatic lung and metastatic breast cancers.
  • MPM malignant pleural mesothelioma
  • Eight monotherapy dose escalation cohorts consisted of 3 patients each (unless otherwise noted): 1.3*10 5 , 2.3x 10’, 3.1 x l0 6 , 4.3xl0 6 , 5.6x l0 6 , 6.1 xl0 7 (6 patients), 7.3xl0 7 , and 8.6x l0 7 cells/kg. All patients except the first 3 received a single dose of cyclophosphamide (CYC) preconditioning (1500 mg/m 2 ). From these 24 CYC + CAR T cell- treated patients, 23 had MPM, 18 of whom were treated with pembrolizumab 200 mg Q3W for a minimum 3 doses, with > 3 months of follow-up after the 3rd dose.
  • CYC cyclophosphamide
  • TEAEs are listed in descending order in the proportion of patients with Grade 1 TEAEs.
  • TEAE treatment-emergent adverse event.
  • an anti-BTN3A monoclonal antibody which activates y982 T cells is also being investigated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors (i.e., EVICTION trial).
  • mAb monoclonal antibody
  • the mAb/yST cell + pembrolizumab treatment combination arm has been well-tolerated during dose escalation (SITC 36th annual meeting December 2021).
  • Dose levels used in preclinical pharmacology studies in mice are determined empirically and typically ranged from 2.5* 10 7 to 6xl0 8 TAC T cells per kg (c/kg), assuming a mouse body weight of 20 g for various TAC T cell products.
  • the Maximum Feasible Dose (MFD) was ty pically 3-fold higher (e.g., 6x l0 8 c/kg).
  • Human equivalent dose levels are calculated based on a direct c/kg conversion and, separately, on body surface area. These calculations indicate the proposed clinical monotherapy starting dose (i.e., Dose Level 1) is approximately 2-3 logs lower than dose levels used in mice.
  • DLTs dose limiting toxicities
  • the exploratory objective of the study is to explore biomarkers that may predict pharmacologic activity or response to TAC T cell monotherapy and when used in combination with pembrolizumab, with endpoints being:
  • Table 9 Summary of Combination Therapy Dose Levels a. Unless the specific disease indication recommends a different dosage.
  • HER2 + tumor types allowed in Phase 1 include, but are not limited to, salivary gland, breast, stomach, ovary, uterus, cervix, lung, biliary tract, pancreas, colorectum, bladder, and prostate.
  • HER2 expression levels of 1+, 2+, and 3+ by IHC are allowed in Phase 1.
  • Phase 2 the following dose expansion groups further evaluate the safety, efficacy, and PK of TAC T cell monotherapy and TAC T cells + pembrolizumab combination therapy at the RP2Ds in 7 prespecified tumor types: breast, lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others.
  • the investigations of these proposed indications are subject to change based on preliminary data obtained in Phase 1, i.e., some may be deleted and others added.
  • TAC T cells Upon enrollment, patients undergo leukapheresis to obtain T cells for the manufacture of TAC T cells. Patients may receive bridging anticancer therapy, after leukapheresis and before lymphodepleting chemotherapy, if deemed necessary by the Investigator. Bridging therapies must be discontinued at least 14 days prior to initiation of lymphodepletion, patients must continue to meet eligibility criteria pertaining to adequate organ function (except hematologic parameters), active infections, pregnancy, measurable disease confirmed by imaging and medication washout before initiation of lymphodepletion. If TAC T cells cannot be manufactured from the first leukapheresis product, additional leukapheresis may be allowed after consultation with the Sponsor.
  • TAC T cells Upon the successful manufacture of TAC T cells, patients enter the treatment phase. It includes lymphodepleting chemotherapy, followed by a single dose of TAC T cells administered intravenously (IV) approximately 48 hours ( ⁇ 24 hours) after completion of lymphodepleting chemotherapy, unless clinical or logistical circumstances require modification of this timing. Any dose adjustments to the lymphodepleting chemotherapy must be discussed in advance with the Medical Monitor. A post-treatment tumor biopsy (in patients with accessible disease) is obtained approximately 8 days following the TAC T cell dose.
  • Radiographic disease assessments (following RECIST 1.1 criteria) are performed pre-treatment, at 1 month, then every 8 weeks (Q8W) up to 24 months following the TAC T cells dose administration.
  • the most recent archival tissue available is an acceptable alternative regardless of when it was obtained during prior treatment or lines of therapy.
  • Tumor samples are screened centrally for HER2 status by both fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) assays. If a patient’s fresh or archival tumor sample can be obtained for central confirmation and is HER2 + on local laboratory results by one of the following 3 methods: IHC, FISH, orNGS, screening and enrollment may proceed, and the central confirmation can be completed after the patient has enrolled in the trial
  • ECOG Eastern Cooperative Oncology Group
  • Colony stimulating factors including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, and other hematopoietic cytokines, within 14 days prior to leukapheresis
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • erythropoietin erythropoietin
  • other hematopoietic cytokines within 14 days prior to leukapheresis
  • CNS central nervous system
  • Active inflammatory neurological disorders e.g., Guillain-Barre Syndrome, amyotrophic lateral sclerosis, multiple sclerosis.
  • Active autoimmune disease e.g, lupus, rheumatoid arthritis, Sjogren’s syndrome
  • Replacement therapy e.g. , thyroxine, insulin, or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.
  • Active hepatitis B or C HCV RNA positive infection or any history of or active human immunodeficiency virus (HIV) infection.
  • Class III or IV heart failure (as defined by the New York Heart Association - NYHA), cardiac angioplasty or stenting, myocardial infarction, unstable angina, or other clinically significant cardiac disease within 6 months prior to screening.
  • Females physiologically capable of becoming pregnant must have a negative serum beta human chorionic gonadotropin (P-hCG) pregnancy test result at screening and within 48 hours prior to the first dose of lymphodepleting chemotherapy.
  • P-hCG human chorionic gonadotropin
  • the dose finding part of this trial utilizes the keyboard design to determine the safety and tolerability of various TAC T cell monotherapy dose levels and used in combination with pembrolizumab.
  • the first 3 trial-treated patients are administered TAC T cells at Dose Level 1 (1 to 3* 10 5 cells/kg).
  • Dose Level 1 (1 to 3* 10 5 cells/kg).
  • treatment of the 2 nd patient must occur > 28 days after the first patient’s infusion.
  • Treatment of the 3 rd patient must occur > 14 days after the 2 nd patient’s infusion.
  • subsequent patients within this dose cohort may be treated without staggering.
  • the interval between treatment of the 1 st and 2 nd patient must also be staggered by at least 28 days.
  • TAC T cells may be treated with TAC T cells in any dose cohort per day.
  • dosing can begin in the combination arm using the previously tested and reviewed TAC T cell dose level(s).
  • NCI National Cancer Institute
  • CCAE Common Terminology Criteria for Adverse Events
  • ASTCT ASTCT Consensus Grading
  • a DLT is defined as:
  • Grade 4 laboratory abnormalities such as electrolyte abnormalities
  • DLT is defined as toxicity that is possibly, probably, or definitely related to studytherapy and may result in a change in the given dose. DLTs include
  • DLTs are assessed from the time of the TAC T cell dose until 28 days following the TAC T cell dose, and up to 42 days should clinically consequential severe neutropenia or thrombocytopenia occur.
  • DLTs are assessed during the first 7 weeks of treatment (i.e., after 2 cycles of pembrolizumab starting on Day 7) during dose escalation and for the first 4 weeks (i.e., after 1 cycle of pembrolizumab starting on Day 7) during dose expansion.
  • the HER2-TAC T cell product is a suspension of genetically modified autologous T cells expressing HER2-TAC (SEQ ID NO: 36) for infusion (i.e., HER2-targeted TAC T cells) containing 10% dimethyl sulfoxide (DMSO) in approximately 20 mL.
  • SEQ ID NO: 36 genetically modified autologous T cells expressing HER2-TAC (SEQ ID NO: 36) for infusion
  • DMSO dimethyl sulfoxide
  • TAC T cells are administered as a single IV infusion approximately 48 hours after completion of lymphodepleting chemotherapy.
  • Lymphodepleting chemotherapy consists of 3 consecutive days of fludarabine (Flu) IV (30 mg/m 2 ) and cyclophosphamide (Cy) IV (300 mg/m 2 ) with or without Mesna IV.
  • Flu fludarabine
  • Cy cyclophosphamide
  • Central venous access is recommended for the infusion of TAC T cells.
  • the patient’s identity must match the patient identifiers on the HER2-TAC T cell product bag.
  • Pembrolizumab 200 mg I.V. is administered every' 3 weeks starting on Day 7 from administration of the TAC-T cells. Dose modifications will follow the guidance described in the approved current USPI.
  • Adverse events are assessed throughout the study according to CTCAE Criteria Version 5,4 Lee 20143 for CRS and ASTCT5 Consensus Criteria. Adverse events, serious adverse events (SAEs), and laboratory abnormalities (type, frequency, and severity) are collected.
  • TAC T cell related toxicities include infusion reactions, CRS, neurotoxicity, macrophage activation syndrome, and tumor lysis syndrome (TLS); the list of these risks may be updated during the study based on observed safety signals.
  • Replication-competent lentivirus (RCL) and if positive, follow-on viral vector sequence testing is performed at specified timepoints during the trial using polymerase chain reaction (PCR)-based assays.
  • PCR polymerase chain reaction
  • Potential pembrolizumab related toxicities include fatigue, musculoskeletal pain, rash, diarrhea, pyrexia, cough, decreased appetite, pruritus, dyspnea, constipation, pain, abdominal pain, nausea, and hypothyroidism.
  • Infusion related reactions and Immune-Mediated Adverse Reactions which may be severe or fatal, can occur in any organ system or tissue for which monitoring for early identification and management is required per current package insert.
  • PK Assessments [0255] Assessment of TAC T cell expansion and persistence in blood (and if available also on cerebrospinal fluid [CSF] and bone marrow [BM] samples) is determined by qPCR to detect the HER2-TAC transgene.
  • Biomarker assessments are performed to evaluate HER2+ tumor status, and immune system characteristics that may be associated with TAC T cell toxicity, efficacy, and resistance mechanisms to TAC T cell treatment.
  • the keyboard dose escalation design governs the number of patients to be enrolled in Phase 1. It is estimated up to 60 patients may be enrolled following the design, i.e., ⁇ 6 patients for each of the 5 dose levels across 2 arms.
  • Treatment with TAC T cells is evaluated both as a monotherapy, and in combination with pembrolizumab, in patients with 7 different HER2 + cancers.
  • This “basket” of tumor types will include lung, pancreatic, colorectal, gastric, endometrial, ovarian, and all others.
  • Each treatment arm enrolls up to 35 total basket cancer patients, with a target of at least 5 patients per tumor type. Rapidly assessing potential efficacy signals across a wide range of cancer indications, using the minimum number of patients, is the goal of this “basket” strategy.
  • the primary efficacy analysis is based on all patients who have measurable disease at the last disease assessment, prior to initiation of study treatments (i.e., TAC T cell monotherapy or TAC T cell + pembrolizumab combination therapy) and who receive study products at the RP2Ds
  • TAC T cells The cellular kinetics of TAC T cells are determined from individual concentration time profiles of circulating TAC T cells and characterized in peripheral blood and summarized by treatment arm.

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Abstract

L'invention concerne des méthodes de traitement du cancer chez des sujets en ayant besoin par l'administration d'une polythérapie comprenant (a) un lymphocyte T exprimant un polypeptide coupleur d'antigène de lymphocytes T (TAC) ayant (i) un domaine de liaison à l'antigène qui se lie à un antigène cible, (ii) un domaine de liaison à l'antigène qui se lie à une protéine associée à un complexe TCR, et (iii) un polypeptide de domaine de signalisation de récepteur de lymphocytes T ; et (b) un inhibiteur de point de contrôle immunitaire.
PCT/US2023/020709 2022-05-02 2023-05-02 Polythérapies comprenant des coupleurs d'antigènes de lymphocytes t et des inhibiteurs de points de contrôle pour le traitement du cancer WO2023215302A2 (fr)

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