WO2024020429A1 - Immune cell therapy - Google Patents

Immune cell therapy Download PDF

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Publication number
WO2024020429A1
WO2024020429A1 PCT/US2023/070469 US2023070469W WO2024020429A1 WO 2024020429 A1 WO2024020429 A1 WO 2024020429A1 US 2023070469 W US2023070469 W US 2023070469W WO 2024020429 A1 WO2024020429 A1 WO 2024020429A1
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Prior art keywords
amino acid
seq
acid sequence
gpc3
catcr
Prior art date
Application number
PCT/US2023/070469
Other languages
French (fr)
Inventor
Guangyan XIONG
Shaohua Xu
Hongruo YUN
Blythe D. SATHER
Bijan A. BOLDAJIPOUR
Rachel C. LYNN
Shobha POTLURI
Hajime Hiraragi
Ying Wang
Original Assignee
Lyell Immunopharma, Inc.
Eureka Therapeutics, Inc.
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Application filed by Lyell Immunopharma, Inc., Eureka Therapeutics, Inc. filed Critical Lyell Immunopharma, Inc.
Publication of WO2024020429A1 publication Critical patent/WO2024020429A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • 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
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/53—Liver
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001174—Proteoglycans, e.g. glypican, brevican or CSPG4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

  • T cell therapy which aims to treat cancer by transferring autologous and ex vivo expanded T cells to patients, has resulted in some notable antitumor responses (Blattman et al., Science. (2004) 305(5681):200-5).
  • TILs tumor infiltrating lymphocytes
  • TIL therapy A major limitation to the widespread application of TIL therapy is the difficulty in generating human T cells with antitumor potential.
  • exogenous high-affinity TCRs can be introduced into normal autologous T cells of the patients through T cell engineering.
  • the adoptive transfer of these cells into lympho-depleted patients has been shown to mediate tumor regression in cancers such as melanoma, colorectal carcinoma, and synovial sarcoma (Kunert et al., Front Immunol. (2013) 4:363; Robbins et al., Clin Cancer Res. (2015) 21(5): 1019-27).
  • TCR-engineered T cell therapy can target the entire array of potential intracellular tumor-specific proteins, which are processed and delivered to the cell surface through MHC presentation; these antigens can be recognized even at a low density by antigen-specific cytotoxic T cells (Kunert, supra). [0005] Attempts have been made to engineer TCR molecules having antibody specificity with T cell receptor effector functions.
  • variable and constant domains of a TCR are replaced by the variable and constant domains of an antibody against a tumor antigen, creating a chimeric antibody-TCR called “abTCR” or “caTCR.”
  • abTCR chimeric antibody-TCR
  • a chimeric stimulating receptor CSR is employed in combination with a caTCR to enhance the tumor-killing efficacy of the engineered T cells.
  • a CSR has an extracellular domain that binds a target ligand, e.g., a tumor antigen, and an intracellular co-stimulatory domain, but unlike a CAR, the CSR does not have an intracellular primary immune cell signaling domain (which typically is a CD3 zeta chain’s intracellular domain) or a functional primary immune cell signaling domain.
  • the CSR and the caTCR may bind to different epitopes of the same target/antigen and work synergistically to boost the cytotoxicity of the engineered T cells. See, e.g., WO 2018/200583, the disclosure of which is incorporated by reference herein in its entirety.
  • T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., reduced cytokine secretion and cytotoxicity), expression of multiple surface inhibitory receptors, and apoptosis.
  • T cell exhaustion has been attributed to constant antigen exposure, leading to continuous TCR signaling, or to tonic antigen-independent signaling through an engineered antigen receptor on T cells (see, e.g., Long, supra).
  • Prevention or reversal of T cell exhaustion has been sought as a means to enhance T cell effectiveness, e.g., in patients with cancer or chronic infections and in T cell therapy. See, e.g., WO 2019/118902, the disclosure of which is incorporated by reference herein in its entirety.
  • Glypican 3 (GPC3, also known as SGB, DGSX, MXR7, SDYS, SGBS, OCL5, SGBS1, GTR2-2) is a cell surface protein that is overexpressed in multiple cancer types, including many solid tumors, such as hepatocellular carcinoma (HCC), melanoma (Nakatsura T et al., Clin Cancer Res. (2004) 10(19) : 6612-21 ), lung squamous cell carcinoma (Yu X et al., Genet Mol Res. (2015) 14(3): 10185-92), ovarian carcinoma (Stadlmann S et al, IntJ Gynecol Pathol.
  • HCC hepatocellular carcinoma
  • melanoma Nakatsura T et al., Clin Cancer Res. (2004) 10(19) : 6612-21
  • lung squamous cell carcinoma Yu X et al., Genet Mol Res. (2015) 14(3): 10185-92
  • compositions and methods for improving immune cell therapy comprising one or more expression cassettes for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that
  • the c-Jun is a wildtype human c-Jun. In some embodiments, the wildtype human c-Jun comprises at least about 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the c-Jun is a mutant human c-Jun. In some embodiments, mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain. In some embodiments, the c-Jun comprises: (i) S63 A and S73 A substitutions as compared to SEQ ID NO: 1; or, (ii) a deletion between amino acid residues 2 and 102 or between amino acid residues 30 and 50 as compared to SEQ ID NO: 1.
  • the GPC3 is a cell-surface bound GPC3.
  • the TCRM is derived from a human y/8 TCR.
  • the caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the caTCR.
  • the stabilization module is selected from the group consisting of a TCR constant domain, a CH1-CL module, a CH2-CH2 module, a CH3- CH3 module, and a CH4-CH4 module.
  • the stabilization module is derived from a human protein.
  • the CL contained in the CH1-CL module is derived from a kappa antibody light chain or lambda antibody light chain.
  • the antigen-binding module of the caTCR is a Fab, a Fab', a (Fab')2, an Fv, or a single chain Fv (scFv).
  • the antigen-binding module of the caTCR comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC- CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15,
  • the antigen-binding module of the caTCR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90%
  • the antigen-binding module of caTCR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR 5 chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR y chain.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR y chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR 5 chain.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 31.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 5.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 63, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 64.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 65, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 66.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 67, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 68.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 69, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 70.
  • the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30, CD28, CD3s, CD3 ⁇ CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.
  • the co-stimulatory immune cell signaling module is derived from the intracellular domain of a co-stimulatory receptor of a TCR.
  • the co-stimulatory receptor is selected from the group consisting of CD30, CD28, 4-IBB, 0X40, ICOS, CD27, and CD40.
  • the co-stimulatory immune cell signaling module of the CSR is derived from human CD30.
  • the co- stimulatory immune cell signaling module of human CD30 comprises the amino acid sequence of SEQ ID NO: 43.
  • the co-stimulatory immune cell signaling module of human CD30 comprises the amino acid sequence of SEQ ID NO: 44.
  • the co-stimulatory immune cell signaling module and the transmembrane domain of the CSR are both derived from CD30 (e.g., human CD30).
  • the ligand-binding module of the CSR comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15,
  • the ligand-binding module of the CSR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90%
  • the ligand-binding module of the CSR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the ligand-binding module CSR comprises: (i) SEQ ID NO: 32, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 32; (ii) SEQ ID NO: 33, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 33; or, (iii) SEQ ID NO: 34, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 34.
  • the antigen-binding module of the caTCR comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and (ii) the ligand-binding module of the CSR comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14
  • the antigen-binding module of the caTCR comprises a Fab that binds to GPC3, wherein the ligand-binding module of the CSR comprises a scFv that binds to GPC3, and wherein the transmembrane module and co-stimulatory immune cell signaling module of the CSR are both derived from CD30 (e.g., human CD30).
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises the amino acid sequence of SEQ ID NO: 32.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29
  • the scFv comprises the amino acid sequence of SEQ ID NO: 34.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27
  • the scFv comprises the amino acid sequence of SEQ ID NO: 32.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25
  • the scFv comprises the amino acid sequence of SEQ ID NO: 34.
  • the construct(s) are viral vectors.
  • the viral vector(s) are selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein- Barr viral vectors.
  • the expression construct comprises a polycistronic expression cassette for expressing the caTCR, the CSR, and the c-Jun.
  • a polycistronic expression construct comprising an expression cassette for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c- Jun polypeptide.
  • TCR chimeric antibody-T cell receptor
  • GPC3 glypican 3
  • TCRM TCR module
  • CSR chimeric stimulating receptor
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 34.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 34.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 34.
  • the coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES).
  • the expression cassette comprises a constitutive or inducible promoter.
  • the promoter is an EF-la promoter.
  • the construct is a viral vector.
  • the viral vector is selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.
  • a recombinant virus comprising the polycistronic expression construct of any of the preceding embodiments.
  • the polycistronic expression construct is a lentiviral vector.
  • a method of engineering immune cells comprising: (a) providing a starting cell population, (b) introducing the expression construct(s) of any one of the preceding claims, or the recombinant virus of any one of the preceding claims into the starting cell population, (c) optionally selecting cells that express the caTCR, the CSR, and the c-Jun, and (d) deriving engineered immune cells from the cells of step (b) or (c).
  • the immune cell is a T cell.
  • the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the starting cell population comprises immune cells.
  • the starting cell population comprises autologous or allogeneic T cells.
  • the starting cell population comprises pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells.
  • a population of human cells comprising the expression construct(s) of any of the preceding embodiments or the recombinant virus of any of the preceding embodiments.
  • the human cells are immune cells.
  • the population of immune cells are obtained by the method of any of the preceding embodiments.
  • the immune cells are human cells.
  • the immune cells are T cells.
  • the T cells are CD8 + T cells.
  • the immune cells express a lower level of an exhaustion marker compared to corresponding cells that do not overexpress c-Jun.
  • the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.
  • a pharmaceutical composition comprising the expression con struct! s) of any of the preceding embodiments, the recombinant virus of any of the preceding embodiments, or the cells of any of the preceding embodiments, and a pharmaceutically acceptable carrier.
  • a method of killing target cells comprising contacting the target cells with the pharmaceutical composition of any of the preceding embodiments under conditions that allow killing of the target cells by the immune cells, wherein the target cells express the GPC3.
  • the immune cells express a lower level of an exhaustion marker when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression.
  • the immune cells are T cells.
  • the T cells are CD8 + T cells.
  • the target cells are cancer cells.
  • the co-stimulatory immune cell signaling module in the CSR is derived from human CD30 and the immune cells express a lower level of an exhaustion marker, as compared to corresponding immune cells engineered to express a CSR whose costimulatory immune cell signaling module is derived from human CD28.
  • the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.
  • provided herein is a method of treating an individual in need thereof, comprising administering the pharmaceutical composition of any of the preceding embodiments to the individual.
  • the individual has a GPC3-positive disease.
  • the GPC3-positive disease is cancer.
  • the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • HCC hepatocellular carcinoma
  • melanoma lung cancer
  • non-small cell lung cancer pancreatic cancer
  • breast cancer triple negative breast cancer
  • lung squamous cell carcinoma ovarian carcinoma
  • yolk sac tumor choriocarcinoma
  • neuroblastoma neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor testicular
  • a method of reducing exhaustion of an engineered immune cell comprising introducing to the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises the expression construct s) of any one of the preceding embodiments.
  • FIG. 1A is a schematic representation of an exemplary pair of caTCR and CSR (the top panel) and the two expression constructs used in in vivo efficacy studies of T cells described herein (the bottom panel).
  • Construct 1 encodes an anti-GPC3 caTCR (“anti-GPC3- caTCR”) plus an anti-GPC3 CSR comprising a co-stimulatory domain derived from CD30 (“anti-GPC3-CD30-CSR”)
  • Construct 2 encodes “anti-GPC3-caTCR”+“anti-GPC3- CD30-CSR”, and a“c-Jun” polypeptide.
  • FIG. IB is a schematic representation of the design of in vivo efficacy studies of T cells expressing anti-GPC3 -caTCR and anti-GPC3-CD30-CSR, with or without c-Jun overexpression, in two different mouse models.
  • FIG. 2 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression, in HepG2-luc mouse model.
  • the T cells were isolated from two healthy human donors. For each T cell type, two doses were tested (5* 10 6 and 2* 10 6 ).
  • FIG. 3 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression in HepG2-luc mouse model in tumor re-challenge studies, wherein the animals were implanted with tumor cells again after the initial tumors regressed.
  • FIGs. 4A-4C are graphs showing peripheral blood T cell counts in HepG2-luc and Hep3B mice injected with anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression.
  • the T cells were isolated from Donor A and the mouse model was HepG2-luc
  • the T cells were isolated from Donor B and the mouse model was HepG2-luc
  • the T cells were isolated from Donor C and the mouse model was Hep3B.
  • FIG. 5 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells, with or without c-Jun overexpression, in Hep3B mouse model.
  • the present application provides engineered human cells (e.g., immune cells such as T cells) comprising constructs for expressing a chimeric antibody-T cell receptor (caTCR), a chimeric stimulating receptor (CSR; also termed “chimeric signaling receptor” herein), and a c-Jun polypeptide (e.g., a human c-Jun polypeptide).
  • a chimeric antibody-T cell receptor e.g., a chimeric stimulating receptor (CSR; also termed “chimeric signaling receptor” herein
  • CSR chimeric signaling receptor
  • c-Jun polypeptide e.g., a human c-Jun polypeptide.
  • GPC3 also referred to as “anti-GPC3 caTCR” and “anti- GPC3 CSR”, respectively, herein).
  • the anti-GPC3 caTCR comprises an antigen-binding module that specifically binds to GPC3, and a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling molecule.
  • the anti-GPC3 CSR comprises a ligand-binding domain that specifically binds to GPC3, and a co-stimulatory immune cell signaling domain capable of providing a stimulatory signal to the immune cell and does not comprise a functional primary T cell signaling sequence.
  • the GPC3 targeted by the anti- GPC3 caTCRs and anti-GPC3 CSRs provided herein may be expressed on the cell surface of a target cell (e.g., a diseased cell).
  • the anti-GPC3 caTCR and the anti- GPC3 CSR bind to different regions on the same GPC3 protein.
  • the disease may be a cancer such as a cancer of the gastrointestinal system, for example, gastric or liver cancer.
  • the anti- GPC3 caTCR is regulated by the naturally occurring machinery that controls TCR activation, while the anti-GPC3 CSR potentiates the immune response mediated by the anti-GPC3 caTCR.
  • c-Jun expression helps sustain the active state of the T cells by, e.g., alleviating or preventing T cell exhaustion.
  • the present engineered immune cells exhibit sustained, potent cytotoxicity against target-bearing tumor cells.
  • T cells that do not overexpress c-Jun (e.g., through an exogenously introduced nucleic acid sequence encoding c-Jun)
  • the present engineered T cells display fewer signs of T cell exhaustion.
  • the engineered cells may have one or more of the following characteristics: (i) they do not have increased expression of exhaustion markers PD-1, TIM-3 and/or LAG-3 over time, (ii) have reduced rates of apoptosis, (iii) they have increased memory cell formation and/or maintenance of memory markers (e.g., CCR7 and CD45RA); (iv) they have enhanced cytotoxicity; (v) they display increased recognition of tumor targets with low surface antigen; (vi) they have enhanced proliferation in response to antigen; (vii) maintain survival and functionality after repeated antigen stimulation; and (vii) they display increased tumorinfiltrating abilities.
  • memory markers e.g., CCR7 and CD45RA
  • the anti-GPC3 CSR comprises a co-stimulatory immune cell signaling domain derived from the intracellular domain of CD30 (e.g., human CD30).
  • CD30 e.g., human CD30.
  • the present inventors have unexpectedly discovered that c-Jun overexpression significantly reduces exhaustion in T cells engineered to express anti-GPC3 caTCR and a CD30-based anti-GPC3 CSR.
  • antibody or “antibody moiety” includes full-length antibodies and antigen-binding fragments thereof.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions (ie., variable domains) of the light and heavy chains are responsible for antigen-binding.
  • the variables region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions ofKabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 5, a, y, and p heavy chains, respectively.
  • IgGl yl heavy chain
  • lgG2 y2 heavy chain
  • lgG3 y3 heavy chain
  • lgG4 y4 heavy chain
  • IgAl al heavy chain
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv 1 ), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • the term “specifically binds” or “is specific for” refers to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody moiety that specifically binds to a target is an antibody moiety that binds the target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets.
  • an antibody moiety that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (for example a cell surface antigen or a peptide/MHC protein complex) with a binding affinity that is at least about 10 times its binding affinity for other targets.
  • one or more antigenic determinants of the antigen for example a cell surface antigen or a peptide/MHC protein complex
  • T cell receptor refers to a heterodimeric receptor composed of aP or y5 chains that pair on the surface of a T cell.
  • Each a, P, y, and 5 chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR), followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region.
  • V variable domain
  • CDR complementarity determining regions
  • C constant domain
  • TM transmembrane
  • the TM region associates with the invariant subunits of the CD3 signaling apparatus.
  • Each of the V domains has three CDRs.
  • CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523- 544; Murphy (2012), xix, 868 p.).
  • pMHC major histocompatibility complex
  • TCR-associated signaling molecule refers to a molecule having a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) that is part of the TCR- CD3 complex.
  • TCR-associated signaling molecules include CD3ys, CD35s, and ( (also known as CD3( ⁇ or CD3( ).
  • Activation refers to the state of the cell that has been sufficiently stimulated to induce a detectable increase in downstream effector functions of the CD3 signaling pathway, including, without limitation, cellular proliferation and cytokine production.
  • module when referring to a portion of a protein is meant to include structurally and/or functionally related portions of one or more polypeptides which make up the protein.
  • a transmembrane module of a dimeric receptor may refer to the portions of each polypeptide chain of the receptor that span the membrane.
  • a module may also refer to related portions of a single polypeptide chain.
  • a transmembrane module of a monomeric receptor may refer to portions of the single polypeptide chain of the receptor that span the membrane.
  • a module may also include only a single portion of a polypeptide.
  • isolated nucleic acid as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • VH CDR3 95-102 96-101 93-101 105-117 109-137
  • VL CDR3 89-97 91-96 89-96 105-117 109-137
  • Residue numbering follows the nomenclature of Kabat et al., supra 2 Residue numbering follows the nomenclature of Chothia et al., supra 3 Residue numbering follows the nomenclature of MacCallum et al., supra 4 Residue numbering follows the nomenclature of Lefranc et al., supra 5 Residue numbering follows the nomenclature of Honegger and Pltickthun, supra
  • chimeric antibodies refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • synthetic in reference to an antibody or antibody moiety means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.
  • Fv is the minimum antibody fragment which contains a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • scFv see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
  • “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • “Homology” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are “homologous” at that position.
  • the “percent of homology” or “percent sequence identity” between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100, considering any conservative substitutions as part of the sequence identity.
  • the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology.
  • a comparison is made when two sequences are aligned to give maximum homology. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792- 1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004).
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • inducible promoter refers to a promoter whose activity can be regulated by adding or removing one or more specific signals.
  • an inducible promoter may activate transcription of an operably linked nucleic acid under a specific set of conditions, e.g., in the presence of an inducing agent or conditions that activates the promoter and/or relieves repression of the promoter.
  • cell engineering or “cell modification” (including derivatives thereof) refers to the targeted modification of a cell, e.g., an immune cell disclosed herein.
  • treatment or “treating” is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of the disease (such as, for example, tumor volume in cancer).
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • recurrence refers to the return of a cancer or disease after clinical assessment of the disappearance of disease. A diagnosis of distant metastasis or local recurrence can be considered a relapse.
  • the term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment.
  • an “effective amount” of a caTCR or composition comprising a caTCR as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and by known methods relating to the stated purpose.
  • the term “therapeutically effective amount” refers to an amount of a caTCR or composition comprising a caTCR as disclosed herein, effective to “treat” a disease or disorder in an individual.
  • the therapeutically effective amount of a caTCR or composition comprising a caTCR as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • a caTCR or composition comprising a caTCR as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.
  • the therapeutically effective amount is a growth inhibitory amount.
  • the therapeutically effective amount is an amount that improves progression free survival of a patient.
  • infectious disease such as viral infection
  • the therapeutically effective amount of a caTCR or composition comprising a caTCR as disclosed herein can reduce the number of cells infected by the pathogen; reduce the production or release of pathogen- derived antigens; inhibit (z.e., slow to some extent and preferably stop) spread of the pathogen to uninfected cells; and/or relieve to some extent one or more symptoms associated with the infection.
  • the therapeutically effective amount is an amount that extends the survival of a patient.
  • pharmaceutically acceptable or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
  • Reference to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X” includes description of "X”.
  • reference to "not" a value or parameter generally means and describes "other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
  • the present invention provides one or more expression constructs comprising one or more expression cassettes for expressing a caTCR (e.g., a caTCR comprising an antigenbinding module that specifically binds to GPC3), a CSR (e.g., a CSR comprising a ligandbinding module that is capable of binding or interacting with GPC3), and a c-Jun polypeptide (e.g., a human c-Jun polypeptide), according to any of the caTCRs, CSRs, and c-Jun polypeptides described herein (such expression construct(s) is also referred to herein as a “caTCR+CSR+c-Jun expression construct s)”).
  • a caTCR e.g., a caTCR comprising an antigenbinding module that specifically binds to GPC3
  • a CSR e.g., a CSR comprising a ligandbinding module that is capable of binding or interacting with G
  • caTCRs and CSRs of the present invention specifically target GPC3 (such caTCRs and CSRs are also referred to herein as “anti-GPC3 caTCRs” and “anti-GPC3 CSRs”, respectively). It should be understood that when used to describe expression constructs or expression cassettes, the words “express/expressing” and “encode/encoding” are used interchangeably herein.
  • an anti-GPC3 caTCR comprising: i) an antigen-binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule;
  • an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligandbinding module and the co-stimulatory immune cell
  • the expression con struct! s) are viral vectors.
  • the viral vector(s) are selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.
  • the expression construct(s) express a polycistronic expression cassette for expressing the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun.
  • the TCRM of the anti-GPC3 caTCR is derived from a human y/8 TCR.
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30).
  • the co-stimulatory immune cell signaling module of the anti-GPC3 CSR is derived from CD30 (e.g., human CD30).
  • the human c-Jun polypeptide is a wildtype human c-Jun polypeptide.
  • one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module comprising: (1) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (2) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM
  • the antigen binding module of the anti-GPC3 caTCR comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5.
  • the anti- GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the ligand binding module of the anti-GPC3 CSR comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 33.
  • the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30).
  • the co-stimulatory immune cell signaling module of the anti-GPC3 CSR is derived from CD30 (e.g., human CD30).
  • the human c-Jun polypeptide is wildtype human c-Jun.
  • the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • an anti-GPC3 caTCR comprising: i) an antigen-binding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module comprising a scFv that is capable of binding or interacting with GPC3; ii) a transmembrane module derived from CD30; and iii) a co-stimulatory immune cell signaling module derived from CD30 that is capable of providing a co-stimulatory immune cell signaling module derived from CD30 that is capable of providing a co-stimulatory immune cell signaling module derived from CD
  • the Fab comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 29.
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the anti-GPC3 CSR comprises a fragment of CD30.
  • the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44.
  • the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16.
  • the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • a polycistronic expression construct comprising an expression cassette for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide.
  • an anti-GPC3 caTCR comprising: i) an antigenbinding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) an anti-GPC3
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 33.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and coding sequences for SEQ ID NOs: 33 and 43.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 16. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 16. In some embodiments, the expression cassette further comprises a coding sequence for a signal peptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 2. In some embodiments, coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF- la promoter. In some embodiments, the construct is a viral vector (such as a lentiviral vector).
  • the expression constructs provided herein encodes a chimeric antibody-T cell receptor (caTCR) construct comprising an antigen binding module that specifically binds to GPC3 (also referred to herein as an “anti-GPC3 caTCR”).
  • GPC3 also referred to herein as an “anti-GPC3 caTCR”.
  • anti-GPC3 caTCR can specifically bind to GPC3 and is capable of recruiting at least one TCR-associated signaling molecule (such as CD35s, CD3ys, and/or CQ.
  • the anti-GPC3 caTCR comprises an antigen-binding module that specifically binds to a cell surface-bound GPC3.
  • an anti-GPC3 caTCR comprising a) an antigen-binding module that specifically binds to GPC3, and b) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) derived from one of the transmembrane domains of a naturally occurring TCR (such as an aPTCR or a ySTCR) and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring TCR (such as an aPTCR or a ySTCR), wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule (such as CD36s, CD3ys, and/or i ), and wherein the antigen-binding module is linked to the first and/or second TCRDs.
  • TCR module comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the anti-GPC3 caTCRs described herein may have one or more features described in this section.
  • any of the features for each component of the anti-GPC3 caTCR e.g., antigen-binding module, TCRD, TCR-TM, spacer module, stabilization module, T cell co-stimulation sequences, various linkers, etc.
  • TCRD antigen-binding module
  • TCR-TM spacer module
  • stabilization module T cell co-stimulation sequences, various linkers, etc.
  • the antigen-binding module (such as an antibody moiety) can specifically bind to GPC3 with a) an affinity that is at least about 10 (including for example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity for other molecules; or b) a Kd no more than about 1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its Kd for binding to other molecules.
  • an affinity that is at least about 10 (including for example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity for other molecules
  • a Kd no more than about 1/10 such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100,
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA).
  • Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.
  • SPR surface plasmon resonance
  • KinExA kinetic exclusion assay
  • the stabilization module comprises a fist stabilization domain and a second stabilization domain.
  • the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the stabilization module is located between the antigenbinding module and the TCRM.
  • the stabilization module is derived from an antibody moiety.
  • the stabilization module is derived from an antibody constant domain.
  • the stabilization module is derived from an antibody heavy chain constant domain.
  • antibody heavy chain constant domains contained in the stabilization module are derived from an IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgA (c.g, IgAl or IgA2), IgD, IgM, or IgE heavy chain, optionally human.
  • an antibody heavy chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived.
  • antibody light chain constant domains contained in the stabilization module are derived from a kappa or lambda light chain, optionally human.
  • the stabilization module is derived from an antibody light chain constant domain.
  • an antibody light chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived.
  • the first and/or second stabilization domains comprise one or more modifications that do not substantially alter their binding affinity for each other.
  • the first and/or second stabilization domains comprise one or more modifications that increase their binding affinity for each other and/or introduce a non-naturally occurring disulfide bond.
  • the stabilization module comprises a knob-into-hole modification (see, for example, Carter P. J Immunol Methods.
  • the stabilization module comprises antibody constant domain regions (e.g., CH3 domains) comprising a knob-into-hole modification.
  • the stabilization module comprises antibody constant domain regions modified by electrostatic steering to enhance their association (see, for example, W02006106905 and Gunasekaran K, et al. J Biol Chem. 285:19637-46, 2010).
  • the first and second stabilization domains are linked by a disulfide bond.
  • stabilization domains include an Fc region; a hinge region; a CH2 domain; a CH3 domain; a CH4 domain; a CHI domain or CL domain; a TCR constant domain; a leucine zipper domain (e.g., ajun/fos leucine zipper domain, see, e.g., Kostelney et al, J.
  • a yeast GCN4 leucine zipper domain an isoleucine zipper domain; a dimerizing region of a dimerizing cell-surface receptor (e.g., interleukin-8 receptor (IL-8R); or an integrin heterodimer such as LFA-1 or GPIIIb/IIIa); a dimerizing region of a secreted, dimerizing ligand (e.g., nerve growth factor (NGF), neurotrophin-3 (NT- 3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), or brain-derived neurotrophic factor (BDNF); see, e.g., Arakawa et al, J. Biol. Chem.
  • NNF nerve growth factor
  • NT- 3 neurotrophin-3
  • IL-8 interleukin-8
  • VEGF vascular endothelial growth factor
  • BDNF brain-derived neurotrophic factor
  • a coiled coil dimerization domain see, for example, WO2014152878; Fletcher et al, ACS Synth. Biol. 1 :240-250, 2012; and Thomas et al., J. Am. Chem. Soc. 135(13):5161-5166, 2013; and a polypeptide comprising at least one cysteine residue (e.g., from about one, two, or three to about ten cysteine residues) such that disulfide bond(s) can form between the polypeptide and a second polypeptide comprising at least one cysteine residue.
  • cysteine residue e.g., from about one, two, or three to about ten cysteine residues
  • the TCRM described herein comprises a first T cell receptor domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule.
  • TCR-TMs are naturally occurring.
  • at least one of the TCR-TMs is non-naturally occurring.
  • both of the TCR-TMs are non-naturally occurring.
  • the first TCR-TM is derived from one of the transmembrane domains of a T cell receptor (such as an aP TCR or a y5 TCR) and the second TCR-TM is derived from the other transmembrane domain of the T cell receptor.
  • the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the transmembrane domains of the T cell receptor. Recruitment of TCR-associated signaling molecules can be determined by methods known in the art, such as FACS analysis for TCR-CD3 complex surface expression or co-immunoprecipitation of CD3 subunits with the caTCR.
  • the first TCR-TM of a TCRM described herein comprises, consists essentially of, or consists of the transmembrane domain of the TCR a chain (e.g., GenBank Accession No: CCI73895) or a variant thereof and the second TCR-TM of the TCRM comprises, consists essentially of, or consists of the transmembrane domain of the TCR P chain (e.g., GenBank Accession No: CCI73893) or a variant thereof.
  • the first TCR-TM comprises, consists essentially of, or consists of the transmembrane domain of the TCR 5 chain (e.g., GenBank Accession No: AAQ57272) or a variant thereof and the second TCR-TM comprises, consists essentially of, or consists of the transmembrane domain of the TCR y chain (e.g., GenBank Accession No: AGE91788) or a variant thereof.
  • the first and second TCR-TMs of a TCRM described herein comprise, consist essentially of, or consist of the transmembrane domain of a TCR a chain constant domain (e.g., SEQ ID NO: 35) or a variant thereof and the transmembrane domain of a TCR P chain constant domain (e.g., SEQ ID NO: 36) or a variant thereof, respectively.
  • the first and second TCR-TMs comprise, consist essentially of, or consist of the transmembrane domain of a TCR 5 chain constant domain (e.g., SEQ ID NO: 37) or a variant thereof and the transmembrane domain of a TCR y chain constant domain (e.g., SEQ ID NO: 38) or a variant thereof, respectively.
  • the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 39 and 40, or variants thereof, respectively.
  • the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 41 and 42, or variants thereof, respectively.
  • Variants of the transmembrane domains include, without limitation, transmembrane domains with one or more amino acid substitutions compared to the reference sequence. In some embodiments, a variant transmembrane domain comprises no more than 5 amino acid substitutions compared to the reference sequence.
  • the first TCRD further comprises a first connecting peptide amino-terminal to the transmembrane domain and/or the second TCRD further comprises a second connecting peptide amino-terminal to the transmembrane domain.
  • the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof
  • the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof.
  • the first and/or second connecting peptides comprise, consist essentially of, or consist of all or a portion of the connecting peptide of a TCR a chain constant domain or a variant thereof and all or a portion of the connecting peptide of a TCR P chain constant domain or a variant thereof, respectively.
  • the first and/or second connecting peptides comprise, consist essentially of, or consist of all or a portion of the connecting peptide of a TCR 5 chain constant domain or a variant thereof and all or a portion of the connecting peptide of a TCR y chain constant domain or a variant thereof, respectively.
  • the first TCRD further comprises a first TCR intracellular domain carboxy-terminal to the first TCR-TM and/or the second TCRD further comprises a second TCR intracellular domain carboxy-terminal to the second TCR-TM.
  • the first TCR intracellular domain comprises all or a portion of the intracellular domain of the TCR subunit from which the first TCR-TM is derived, or a variant thereof
  • the second TCR intracellular domain comprises all or a portion of the intracellular domain of the TCR subunit from which the second TCR-TM is derived, or a variant thereof.
  • the first TCRD is a fragment of one chain of a naturally occurring TCR, or a variant thereof
  • the second TCRD is a fragment of the other chain of the naturally occurring TCR, or a variant thereof.
  • at least one of the TCRDs is non-natural occurring.
  • a non-naturally occurring TCR domain may be a corresponding domain of a naturally occurring TCR modified by substitution of one or more amino acids, and/or by replacement of a portion of the corresponding domain with a portion of an analogous domain from another TCR.
  • the first TCR-TM comprises, consists essentially of, or consists of all or a portion of SEQ ID NO: 3, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 3.
  • the second TCR-TM comprises, consists essentially of, or consists of all or a portion of SEQ ID NO: 4, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 4.
  • the first and second TCRDs are linked by a disulfide bond. In some embodiments, the first and second TCRDs are linked by a disulfide bond between a residue in the first connecting peptide and a residue in the second connecting peptide.
  • the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( . In some embodiments, the TCRM is capable of recruiting each of CD35s, CD3ys, and ( to form a caTCR-CD3 complex (z.e., promotes caTCR-CD3 complex formation).
  • the antigen-binding module is an antibody moiety selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
  • the antigen-binding module is an antibody moiety that is a Fab or a Fab’.
  • the antigen-binding module is an antibody moiety that is an scFv.
  • the antibody moiety is fully human, semisynthetic with human antibody framework regions, or humanized.
  • the antigen-binding module comprises a first antigen-binding domain comprising an anti-GPC3 VH antibody domain and a second antigen-binding domain comprising an anti-GPC3 VL antibody domain.
  • the anti-GPC3 VH antibody domain and anti-GPC3 VL antibody domain CDRs are derived from the same antibody moiety.
  • some of the anti-GPC3 VH antibody domain and anti-GPC3 VL antibody domain CDRs are derived from different antibody moieties.
  • the anti-GPC3 VH antibody domain and/or anti-GPC3 VL antibody domain are human, humanized, chimeric, semi -synthetic, or fully synthetic.
  • the antigen-binding module in some embodiments is an antibody moiety comprising specific CDR sequences derived from one or more antibody moieties specific to GPC3 (such as a monoclonal antibody) or certain variants of such sequences comprising one or more amino acid substitutions.
  • the amino acid substitutions in the variant sequences do not substantially reduce the ability of the antigen-binding module to bind to GPC3. Alterations that substantially improve target antigen binding affinity or affect some other property, such as specificity and/or cross-reactivity with related variants of GPC3, are also contemplated.
  • the anti-GPC3 caTCR comprises an antigen-binding module described herein linked to a TCRM described herein, optionally including a stabilization module.
  • the anti-GPC3 caTCR comprises the antigenbinding module linked to the N-terminus of one or both of the TCRDs.
  • the anti-GPC3 caTCR comprises a stabilization module between a TCRM and an antigen-binding module.
  • the anti-GPC3 caTCR further comprises a spacer module between any two anti-GPC3 caTCR modules or domains.
  • the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length.
  • the anti-GPC3 caTCR further comprises one or more accessory intracellular domains.
  • the one or more accessory intracellular domains are carboxy -terminal to the first and/or second TCRD.
  • the one or more accessory intracellular domains are between the first TCR-TM and the first TCR intracellular domain and/or between the second TCR-TM and the second TCR intracellular domain.
  • the one or more accessory intracellular domains comprise, individually, a TCR co-stimulatory domain.
  • the TCR co-stimulatory domain comprises all or a portion of the intracellular domain of an immune co-stimulatory molecule (such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like).
  • an immune co-stimulatory molecule such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like).
  • the anti-GPC3 caTCR described herein comprises a) an antigen-binding module that specifically binds to GPC3, and b) a TCRM comprising first and second TCR-TMs derived from the transmembrane domains of a TCR (such as an aPTCR or a ySTCR), wherein the TCRM is capable of recruiting at least one TCR-associated signaling molecule.
  • the antigen-binding module is linked to the amino-terminus of one or more polypeptide chains in the TCRM.
  • the TCRM comprises two polypeptide chains, and the antigen-binding module is linked to the amino-terminus of one or both of the TCRM polypeptide chains.
  • the first and second TCR-TMs are naturally occurring.
  • at least one of the TCR-TMs is non-naturally occurring.
  • the first and second TCR-TMs are non-naturally occurring.
  • the TCRM further comprises at least one connecting peptide or fragment thereof of the TCR amino-terminal to a TCR-TM.
  • the TCRM further comprises at least one TCR intracellular domain comprising a sequence from an intracellular domain of the TCR carboxy-terminal to a TCR-TM.
  • the TCRM comprises TCRDs derived from fragments of the TCR chains.
  • at least one of the TCRDs is non-naturally occurring.
  • the caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4- IBB (CD137), 0X40, CD30, or CD40) carboxy-terminal to a TCR-TM.
  • the caTCR lacks a co-stimulatory signaling sequence.
  • the antigenbinding module is an antibody moiety that specifically binds to GPC3 (“anti-GPC3 antibody moiety”).
  • the anti-GPC3 antibody moiety comprises an anti-GPC3 VH antibody domain and an anti-GPC3 VL antibody domain.
  • the anti- GPC3 antibody moiety is human, humanized, chimeric, semi-synthetic, or fully synthetic.
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the stabilization module comprises at least one disulfide bond linking the stabilization domains.
  • the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof.
  • the TCRM is capable of recruiting at least one TCR- associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( .
  • the TCRM allows for enhanced recruitment of the at least one TCR- associated signaling molecule as compared to a TCRM comprising the T cell receptor transmembrane domains.
  • the TCRM promotes caTCR-CD3 complex formation.
  • the anti-GPC3 caTCR is a heteromultimer, such as a heterodimer.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD, wherein the antigen-binding module is linked to the first and/or second polypeptide chains.
  • the anti-GPC3 caTCR described herein specifically binds GPC3, and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a TCR and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM that is capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen-binding module that specifically binds to GPC3, wherein the antigen-binding module is linked to the first and/or second TCRDs.
  • both of the TCR-TMs are naturally occurring.
  • the TCR- TMs is non-naturally occurring.
  • the TCR is an aP TCR and the first and second TCR-TMs are derived from TCR a and P subunit transmembrane domains.
  • the TCR is a y5 TCR and the first and second TCR-TMs are derived from TCR y and 5 subunit transmembrane domains.
  • the first TCRD further comprises a first TCR connecting peptide or a fragment thereof and/or the second TCRD further comprises a second TCR connecting peptide or a fragment thereof.
  • the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof
  • the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof.
  • the first and second connecting peptides are linked by a disulfide bond.
  • the first TCRD further comprises a first TCR intracellular domain and/or the second TCRD further comprises a second TCR intracellular domain.
  • the first TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the first TCR-TM is derived and/or the second TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the second TCR-TM is derived.
  • the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived.
  • the anti-GPC3 caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4- 1BB (CD137), 0X40, CD30, or CD40).
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the first and second stabilization domains are linked by a disulfide bond.
  • the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof.
  • the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( .
  • the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the T cell receptor transmembrane domains.
  • the TCRM promotes caTCR-CD3 complex formation.
  • the antigen-binding module is an antibody moiety (e.g., and anti-GPC3 antibody moiety).
  • the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
  • the anti-GPC3 caTCR described herein specifically binds GPC3, and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring y5 TCR and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring y5 TCR, wherein the first and second TCRDs form a TCRM that is capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen-binding module that specifically binds to GPC3a, wherein the antigen-binding module is linked to the first and/or second TCRDs.
  • both of the TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring.
  • the first TCRD further comprises a first TCR connecting peptide or a fragment thereof and/or the second TCRD further comprises a second TCR connecting peptide or a fragment thereof.
  • the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof
  • the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof.
  • the first and second connecting peptides are linked by a disulfide bond.
  • the first TCRD further comprises a first TCR intracellular domain and/or the second TCRD further comprises a second TCR intracellular domain.
  • the first TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the first TCR-TM is derived and/or the second TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the second TCR-TM is derived.
  • the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived.
  • the anti-GPC3 caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), 0X40, CD30, or CD40).
  • the anti- GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the first and second stabilization domains are linked by a disulfide bond.
  • the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof.
  • the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( .
  • the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the naturally occurring y5 T cell receptor transmembrane domains.
  • the TCRM promotes caTCR-CD3 complex formation.
  • the antigen-binding module is an antibody moiety (e.g., and anti-GPC3 antibody moiety).
  • the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN,
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the antigen-binding module is a Fab.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
  • the antigen-binding module is a Fab.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS,
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
  • the anti-GPC3 antibody moiety is a Fab.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 25.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 24; and b) a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the anti- GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 24, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the antigenbinding module is a Fab.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 26, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the antigenbinding module is a Fab.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 29.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 28, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the antigenbinding module is a Fab.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR a chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR P chain.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR P chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR a chain.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR 5 chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR y chain.
  • the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR y chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR 5 chain.
  • fused means directly fused or indirectly fused.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 30; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 31.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 5.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 63; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 64.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 65; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 66.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 67; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 68.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68.
  • the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 69; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 70.
  • anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70.
  • the expression constructs provided herein further encodes a chimeric costimulatory receptor (CSR) that specifically binds to GPC3 (also referred to herein as an “anti-GPC3 CSR”).
  • the anti-GPC3 CSR is capable of stimulating an immune cell on the surface of which it is functionally expressed upon target ligand binding.
  • the anti-GPC3 CSR comprises a ligand-binding module that provides the ligand-binding specificity, a transmembrane module, and a co-stimulatory immune cell signaling module that allows for stimulating the immune cell.
  • the anti-GPC3 CSR lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-GPC3 CSR lacks any primary immune cell signaling sequence.
  • the anti-GPC3 CSR comprises a single polypeptide chain comprising the ligand-binding module, transmembrane module, and costimulatory signaling module. In some embodiments, the anti-GPC3 CSR comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the ligand-binding module, transmembrane module, and co-stimulatory signaling module. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-GPC3 CSR is a multimer, such as a dimer.
  • first and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond.
  • co-stimulatory immune cell signaling domains for use in the anti- GPC3 CSRs of the invention include the cytoplasmic sequences of co-receptors of the T cell receptor (TCR), which can act in concert with a caTCR (e.g., an anti-GPC3 as described herein) to initiate signal transduction following caTCR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation is mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as “primary T cell signaling sequences”) and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (referred to herein as “co-stimulatory T cell signaling sequences”).
  • primary T cell signaling sequences those that initiate antigen-dependent primary activation through the TCR
  • co-stimulatory T cell signaling sequences those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal
  • Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM-containing primary immune cell signaling sequences include those derived from TCR FcRy, FcRp, CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, and CD66d.
  • a “functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor.
  • “Non-functional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal.
  • the anti-GPC3 CSRs described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an ITAM. In some embodiments, the anti-GPC3 CSRs lack any primary immune cell signaling sequence.
  • the co-stimulatory immune cell signaling sequence can be a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4- 1BB (CD137), 0X40, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
  • a co-stimulatory molecule including, for example, CD27, CD28, 4- 1BB (CD137), 0X40, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
  • the target ligand of the anti-GPC3 CSR is GPC3.
  • the GPC3 is a cell surface-bound GPC3.
  • the GPC3 target ligand is the same as the GPC3 target antigen of a caTCR (e.g., anti-GPC3 caTCR as described herein) expressed in the same immune cell.
  • the ligand-binding module in the anti-GPC3 CSR is an antibody moiety that specifically binds GPC3 (“anti-GPC3 antibody moiety).
  • the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
  • the ligand-binding module is derived from the extracellular domain of a receptor.
  • the antibody moiety of the anti-GPC3 CSR comprises the CDRs or variables domains (VH and/or VL domains) of an antibody moiety specific for GPC3.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN
  • the anti-GPC3 CSR comprises a ligand-binding module comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the ligand binding module is a scFv.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of Y
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
  • the ligand binding module is a scFv.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
  • the ligand binding module is a scFv.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 25.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 24; and b) a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 24, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 25.
  • the ligand binding module is a scFv.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the anti- GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 26, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the ligand binding module is a scFv.
  • the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 29.
  • the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the anti- GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 28, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the ligand binding module is a scFv.
  • the transmembrane domain of the anti-GPC3 CSR comprises a transmembrane domain derived from a transmembrane protein including, for example, CD28, CD3s, CD3 ⁇ CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.
  • the CD30 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 49, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 49.
  • the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD28.
  • the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 50.
  • the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD8.
  • the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 22.
  • the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from the intracellular domain of a co-stimulatory receptor of a TCR.
  • the co-stimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of all or a portion of the intracellular domain of a co-stimulatory receptor of, for example, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
  • the co-stimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of all or a portion of the intracellular domain of a co-stimulatory receptor of CD30, CD28, 4-IBB, 0X40, ICOS, CD27, or CD40.
  • the co-stimulatory immune cell signaling module of the CSR is derived from human CD30.
  • the co-stimulatory immune cell signaling module derived from human CD30 comprises the amino acid sequence of SEQ ID NO: 44, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 44.
  • the co-stimulatory immune cell signaling module of the CSR is derived from human CD28.
  • the co-stimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 45, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 45.
  • the co- stimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 46, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 46.
  • the co-stimulatory immune cell signaling module of the CSR is derived from human 4-IBB.
  • the co-stimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 47, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 47.
  • the co-stimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 48, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 48.
  • the anti-GPC3 CSR further comprises a spacer module between any of the ligand-binding module, the transmembrane module, and the co- stimulatory signaling module.
  • the spacer module comprises one or more peptide linkers connecting two anti-GPC3 CSR modules.
  • the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length.
  • the ligand-binding module (such as an antibody moiety) of the anti-GPC3 CSR specifically binds to GPC3 with a) an affinity that may be at least about 10 (including for example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity for other molecules; or b) a Kd the may be more than about 1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its Kd for binding to other molecules.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA).
  • Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.
  • SPR surface plasmon resonance
  • KinExA kinetic exclusion assay
  • the anti-GPC3 CSR described herein comprises a) an scFv; and b) a fragment of CD30.
  • the scFv comprises a VH domain having the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 26, and a VL domain having the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 27.
  • the scFv comprises a VH domain having the amino acid sequence of SEQ ID NO: 26 and a VL domain having the amino acid sequence of SEQ ID NO: 27.
  • the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 33.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44.
  • the CSR comprises, from amino terminus to carboxy terminus, the scFv, a peptide linker, and the fragment of CD30. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16.
  • the expression constructs provided herein further encodes a c-Jun polypeptide.
  • the c-Jun is a human c-Jun.
  • the human c-Jun is wildtype human c-Jun.
  • the human c-Jun has the following amino acid sequence (available at GenBank under accession number AAA59197.1 or at UniProtKB (under accession number P05412.2): MTAKMETTFY DDALNASFLP SESGPYGYSN PKILKQSMTL NLADPVGSLK PHLRAKNSDL LT[S]PDVGLLK LA ⁇ S]PELERLI IQSSNGHITT TPTPTQFLCP KNVTDEQEGF AEGFVRALAE LHSQNTLPSV TSAAQPVNGA GMVAPAVASV AGGSGSGGFS ASLHSEPPVY ANLSNFNPGA LSSGGGAPSY GAAGLAFPAQ PQQQQPPHH LPQQMPVQHP RLQALKEEPQ TVPEMPGETP PLSPIDMESQ ERIKAERKRM RNRIAASKCR KRKLERIARL EEKVKTLKAQ NSELASTANM LREQVAQLKQ KVMNHVNSGC QLMLTQQLQT
  • the c-Jun protein is encoded by the JUN gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047 of GenBank Accession No. NC_000001.11, minus strand orientation).
  • JUN Jun proto-oncogene
  • AP-1 transcription factor subunit v-Jun avian sarcoma virus 17 oncogene homolog
  • transcription factor AP-1 avian sarcoma virus 17 oncogene homolog
  • transcription factor AP-1 transcription factor AP-1
  • Jun oncogene AP-1
  • Jun activation domain binding protein p39
  • Enhancerbinding protein API The wild-type human c-Jun protein sequence is 331 amino acids in length.
  • the wildtype human c-Jun comprises at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the wildtype human c-Jun comprises the amino acid sequence of SEQ ID NO: 1.
  • the c-Jun is a mutant c-Jun. In some embodiments, the c-Jun is a mutant human c-Jun. In some embodiments, the mutant c-Jun does not impact the mutant’s ability to rescue dysfunctional (exhausted) T cells.
  • the mutant c-Jun comprises at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, or 99%) sequence identity with the C-terminal amino acid residues (e.g., C-terminal 50, 75, 100, 150, 200, 250, or more, amino acid residues), the C-terminal portion (e.g., quarter, third, or half) or C-terminal domains (e.g., epsilon, bZIP, and amino acids C-terminal thereof) of a wildtype c-Jun.
  • the C-terminal amino acid residues e.g., C-terminal 50, 75, 100, 150, 200, 250, or more, amino acid residues
  • the C-terminal portion e.g., quarter, third, or half
  • C-terminal domains e.g., epsilon, bZIP, and amino acids C-terminal thereof
  • the N-terminal amino acid residues e.g., N-terminal 50, 75, 100, or 150 or more
  • the N-terminal portion e.g., quarter, third, or half
  • N- terminal domains e.g., delta, transactivation domain, and amino acids N-terminal thereof
  • the c-Jun comprises one or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating substitutions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating deletions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating insertions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its delta domain.
  • inactivating mutation(s) e.g., substitutions, deletions, or insertions
  • the c-Jun comprises one or more inactivating substitutions in its delta domain. In some embodiments, the c-Jun comprises one or more inactivating deletions in its delta domain. In some embodiments, the c-Jun comprises one or more inactivating insertions in its delta domain. In some embodiments, the c-Jun comprises or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its transactivation domain and its delta domain.
  • the c-Jun comprises a serine (S) to alanine (A) substitution.
  • the c-Jun comprises one or both of S63 A and S73 A substitutions as compared to SEQ ID NO: 1 (the positions are boxed in the amino acid sequence of human c- Jun above).
  • the c-Jun has a deletion between amino acid residues 2 and amino acid residues 102 or between residues 30 and 50 as compared to SEQ ID NO: 1.
  • the anti-GPC3 caTCR and/or the anti-GPC3 CSR and/or the c-Jun polypeptides further comprise an affinity or purification tag available for various use purposes; for example, it may be used to enhance the purification efficiency of the target polypeptide.
  • the tag is a myc, HIS or HA tag.
  • an immune cell described herein has been modified to comprise an exogenous nucleotide sequence encoding a c-Jun polypeptide, wherein the exogenous nucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 52-62.
  • an exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 52-62. [0137] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 52.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 52.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 52.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 53.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 53.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 53.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 54.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 54.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 54.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 55.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% to the nucleic acid sequence set forth in SEQ ID NO: 55.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 55.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 56.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 56.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 56.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57.
  • the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 57.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 58.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 58.
  • the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 58.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 59.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 59.
  • the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 59.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 60.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 60.
  • the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 60.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 61.
  • an exogenous polynucleotide encoding a c-Jun polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 61.
  • the exogenous nucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 61.
  • the c-Jun nucleotide sequence disclosed herein can be codon-optimized using any methods known in the art.
  • the codons of a c-Jun nucleotide sequence disclosed herein has been optimized to modify (e.g., increase or decrease) one or more of the following parameters compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 62): (i) codon adaptation index (z.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and unstable motifs; (iv) repeat sequences (e.g., direct repeats, inverted repeats, dyad repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.
  • codon adaptation index z.e., codon usage bias
  • GC guanine-cytosine
  • mRNA secondary structure and unstable motifs e.g., direct repeats, in
  • c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa).
  • CRISPRa a transcriptional activator
  • exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa).
  • transcriptional activator e.g., CRISPRa
  • the engineered cells overexpress, i.e., express a higher level (e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least about 1.5-, 2-, 3-, 4-, 5-, or 10-fold more) of, a c-Iun protein than corresponding cells without such a modification ("reference cell").
  • a higher level e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least about 1.5-, 2-, 3-, 4-, 5-, or 10-fold more
  • the terms “express increased levels [or amounts] of,” “overexpress,” or have “increased expression of' (and similar forms of the phrase used herein) are used interchangeably.
  • the engineered (or modified) cells described herein express at least about 2-100 fold more, about 5-50 fold more, about 5-40 fold more, about 5-30 fold more, about 5-20 fold more, about 8-20 fold more, or about 10-20 fold more c-Iun protein than the reference cell.
  • the expression of the c-Iun protein in a modified cell described herein is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45- fold, or by at least about 50-fold, compared to the expression of the c-Iun protein in the reference cell.
  • the increased expression of the c-Iun protein can improve and/or enhance one or more properties of the modified immune cells (e.g., T cells, such as CD4+ and/or CD8+ T cells).
  • modified immune cells e.g., T cells, such as CD4+ and/or CD8+ T cells.
  • properties include: resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers, such as PD-1, CD39, TIM-3, and/or LAG-3; increased persistence/survival; delay of the onset of dysfunctional states; and/or increased cytokine (e.g., IFN-y and/or IL-2) production), increased expansion/proliferation, increased antigen sensitivity, improved effector function, in particular, improved effector function following repeated antigen stimulation (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or combinations thereof.
  • resistance to exhaustion e.g., as indicated by reduced expression of exhaustion
  • Assays useful for measuring exhaustion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, cytokine production/release, and gene expression profiles are known in the art and include, for example flow cytometry, intracellular cytokine staining (ICS), INCUCYTE® immune cell killing analysis, Meso Scale Discovery (MSD, a platform which combines electrochemiluminescence and MULTI-ARRAY technology) or similar assay, persistent antigen stimulation assays, bulk and single cell RNAseq (see e.g., Front Genet. 2020; 11 :220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci.
  • ICS intracellular cytokine staining
  • MSD Meso Scale Discovery
  • the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be introduced to the T cells or progenitor cells through one or more nucleic acid molecules (e.g., DNA or RNA such as mRNA).
  • the nucleic acid molecules may be placed on one or more DNA or RNA vectors for introduction into the host cells.
  • the nucleic acid molecules may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes).
  • the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules.
  • viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus.
  • the coding sequences for the two polypeptide chains of the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be placed on a single expression construct.
  • the four coding sequences may be placed into one or more expression cassettes on the construct, each cassette being its own transcription unit (e.g., with its own promoter and polyadenylation site and other transcription control elements).
  • the four coding sequences may be placed into a single expression cassette (e.g., a polycistronic expression cassette), with the four coding sequences being transcribed under a common promoter.
  • the coding sequences are in-frame and separated from each other by the coding sequence of a self-cleaving peptide (e.g., a 2A self-cleaving peptide such as a T2A, P2A, E2A, or F2A peptide).
  • a self-cleaving peptide e.g., a 2A self-cleaving peptide such as a T2A, P2A, E2A, or F2A peptide.
  • the coding sequences may be separated from each other by a ribosomal internal entry site (IRES).
  • IRS ribosomal internal entry site
  • the polycistronic expression cassette comprises coding sequences for the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun. In some embodiments, the expression cassette comprises coding sequences for the antigen binding module of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 28 and 29. In some embodiments, the expression cassette comprises coding sequences for a TCRM derived from a human y/8 TCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 4 and 5.
  • the expression cassette comprises coding sequences for the two polypeptide chains of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 30 and 31. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 10 and 5. In some embodiments, the expression cassette comprises a coding sequence for the ligand binding module of the anti-GPC3 CSR. In some embodiments, the expression cassette comprises a coding sequence for the amino acid sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for a partial sequence of human CD30 of the anti-GPC3 CSR.
  • the expression cassette comprises a coding sequence for the amino acid sequence of SEQ ID NO: 43. In some embodiments, the expression cassette comprises a coding sequence for the human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 33.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and coding sequences for SEQ ID NOs: 33 and 43.
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette further comprises a coding sequence for a signal peptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 2.
  • the expression cassettes may contain a promoter that is constitutively active in mammalian (e.g., human or human T) cells.
  • promoters include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor- la (EF-la) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.
  • CMV immediate early cytomegalovirus
  • SV40 simian virus 40
  • HSV40 human immunodeficiency virus
  • LTR long terminal repeat
  • Epstein-Barr virus immediate early promoter a Rous sarcoma virus immediate early promoter
  • the expression cassettes may comprise an inducible promoter system.
  • Exemplary inducible promoter systems include, without limitation, hormone-regulated elements, synthetic ligand- regulated elements, ionizing radiation-regulated elements, tetracycline (Tet) systems (e.g., “Tet-Off” and “Tet-On” systems), and NF AT systems (see, e.g., Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics. (2016) 12: 16-25).
  • the expression cassettes include a signal peptide.
  • the signal peptide comprises the coding sequence for the amino acid sequence of SEQ ID NO: 2.
  • the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and/or translation of the coding sequences.
  • a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3 ’ untranslated region of the expression cassette.
  • the transcription/translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts.
  • the present disclosure provides a single-vector construct (e.g., a lentiviral vector) comprising a polycistronic expression cassette, comprising a mammalian promoter, a c-Jun coding sequence, coding sequences for the two anti-GPC3 caTCR chains, a coding sequence for the anti-GPC3 CSR, and a polyadenylation signal sequence.
  • the coding sequences are linked by a nucleotide linker that may be an IRES or a coding sequence for a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A, or functional equivalents thereof).
  • FIG. 1A illustrates such an expression cassette, wherein the promoter is an EF- la promoter.
  • the expression cassette encodes a c-Jun comprising SEQ ID NO: 1, an anti-GPC3 caTCR comprising two polypeptide chains comprising SEQ ID NOs: 30 and 31, respectively, and an anti-GPC3 CSR comprising SEQ ID NO: 33 (e.g., Construct 2 in FIG. 1A).
  • the construct may be a recombinant lentiviral vector and may further comprise a central polypurine tract (cPPT) upstream of the EF-la promoter, and a WPRE sequence between the CSR coding sequence and an SV40 polyadenylation signal, or other sequences for efficient transduction and expression in mammalian cells.
  • cPPT central polypurine tract
  • the coding sequences in the expression cassettes may be codon-optimized for optimal expression levels in a host cell of interest (e.g., human cells).
  • the nucleic acid molecules encoding the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be integrated into the genome of the engineered cells or remain episomal.
  • the integration may be targeted integration occurring through gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases, and meganucleases).
  • the engineered cells can be enriched for by positive selection techniques.
  • the cells can be selected for their ability to bind to GPC3 in, e.g, flow cytometry assays.
  • RT-PCT may be performed on the engineered T cells.
  • the positive selection may lead to enrichment of caTCR + CSR + c-Jun + cells in a cell population, where the triple positive T cells constitute more than 30, 35, 40, 45 ,50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the total cell population.
  • the engineered cells may be cryopreserved until use.
  • amino acid sequence variants of the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun provided herein are contemplated.
  • amino acid sequence variants of the antigen binding module of the anti-GPC3 caTCR and/or the ligand binding module of the anti-GPC3 CSR are contemplated.
  • Amino acid sequence variants of an anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun, or by peptide synthesis.
  • modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., GPC3-binding.
  • anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs (hypervariable regions) and FRs (framework regions).
  • Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; and f. aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • An exemplary substitutional variant is an affinity-matured antibody moiety comprised of the antigen binding module of the anti-GPC3 caTCR or the ligand binding module of the anti-GPC CSR, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties are displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody moiety affinity.
  • HVR hotspots
  • SDRs specificity determining residues
  • variable genes chosen for maturation are introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody moiety variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the anti- GPC3 caTCR and/or the anti-GPC3 CSR to bind GPC3.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or SDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody e.g., an antibody moiety comprised in the antigen binding module of the anti-GPC3 caTCR or the anti-GPC3 CSR
  • alanine scanning mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085.
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex can be determined to identify contact points between the anti-GPC3 caTCR and/or the anti-GPC3 CSR and GPC3. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they exhibit the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody moiety (e.g, an antigen binding module of the anti- GPC3 caTCR and/or a ligand binding module of the anti-GPC3 CSR) with an N-terminal methionyl residue.
  • insertional variants of the antibody moiety include the fusion to the N- or C-terminus of the antibody moiety to an enzyme (e.g for ADEPT, antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the construct (e.g., the anti-GPC3 caTCR and/or anti-GPC3 CSR construct).
  • an enzyme e.g for ADEPT, antibody-directed enzyme prodrug therapy
  • a polypeptide which increases the serum half-life of the construct e.g., the anti-GPC3 caTCR and/or anti-GPC3 CSR construct.
  • an anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun provided herein is altered to increase or decrease the extent to which the expression construct is glycosylated. Addition or deletion of glycosylation sites to an expression construct may be conveniently accomplished by altering the amino acid sequence of the anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun construct or polypeptide portion thereof such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an anti-GPC3 construct of the invention may be made in order to create anti-GPC3 construct variants with certain improved properties.
  • N-glycans attached to the CH2 domain of Fc are heterogeneous.
  • Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum.
  • N-glycosylation of the Fc is important for binding to FcyR; and afucosylation of the N-glycan increases Fc's binding capacity for FcyRIIIa. Increased FcyRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications in which cytotoxicity is desirable.
  • an enhanced effector function can be detrimental when remediated cytotoxicity is undesirable.
  • the Fc fragment or CH2 domain is not glycosylated.
  • the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.
  • cysteine engineered anti-GPC3 caTCR constructs in which one or more amino acid residues are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the constructs described herein. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the construct and may be used to conjugate the anti-GPC3 construct to other moieties.
  • Cysteine engineered expression constructs may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
  • the present invention in one aspect provides modified cells (such as immune cells, for example T cells) expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a human c-Jun polypeptide, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and a human c-Jun polypeptide described herein.
  • modified cells such as immune cells, for example T cells
  • a human c-Jun polypeptide such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and a human c-Jun polypeptide described herein.
  • the present invention provides an immune cell (such as a T cell) presenting on its surface a caTCR, an CSR, and a c-Jun polypeptide according to any of the caTCRs, CSRs, and c-Jun polypeptides described herein described herein (such an immune cell is also referred to herein as an “caTCR plus CSR plus c-Jun immune cell”).
  • the caTCRs and CSRs of the present invention specifically target GPC3 (such caTCRs and CSRs are also referred to herein as “anti-GPC3 caTCRs” and “anti-GPC3 CSRs”, respectively).
  • the immune cell comprises nucleic acid encoding the anti-GPC3 caTCR and anti-GPC3 CSR, wherein the anti-GPC3 caTCR and anti-GPC3 CSR are expressed from the nucleic acid and localized to the immune cell surface.
  • the immune cell is a T cell.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived.
  • the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the T cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of the endogenous TCR subunits of the immune cell.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or P chains or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • Modifications of cells to disrupt gene expression include any such techniques known in the art, including for example RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR- or TALEN-based gene knockout), and the like.
  • an immune cell (such as a T cell) comprising nucleic acid encoding an anti-GPC3 caTCR according to any of the anti- GPC3 caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptide are expressed from the nucleic acid and localized to the immune cell surface.
  • the nucleic acid comprises a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of the anti-GPC3 caTCR, an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR, and a human c-Jun nucleic acid sequence encoding a human c- Jun polypeptide chain.
  • the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and human c-Jun nucleic acid sequences are each contained in different vectors. In some embodiments, some or all of the nucleic acid sequences are contained in the same vector.
  • Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses). In some embodiments, one or more of the vectors is integrated into the host genome of the immune cell.
  • the first and second anti-GPC3 caTCR nucleic acid sequences, the anti-GPC3 CSR nucleic acid sequence, and the human c-Jun nucleic acid sequence are each under the control of different promoters. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • an anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun (“caTCR+CSR+c-Jun”) immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the anti-GPC3 caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR;
  • the first anti-GPC3 caTCR nucleic acid sequence is contained in a first vector (such as a lentiviral vector)
  • the second anti-GPC3 caTCR nucleic acid sequence is contained in a second vector (such as a lentiviral vector)
  • the anti-GPC3 CSR nucleic acid sequence is contained in a third vector (such as a lentiviral vector)
  • the c-Jun nucleic acid sequences is contained in a fourth vector (such as a lentiviral vector).
  • some or all of the first and second anti-GPC3 caTCR nucleic acid sequences and anti-GPC3 CSR nucleic acid sequence are contained in the same vector (such as a lentiviral vector).
  • first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and c-Jun nucleic acid sequence are contained in the same vector (such as a lentiviral vector).
  • each of the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and c-Jun nucleic acid sequence are, individually, operably linked to a promoter.
  • some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters have the same sequence.
  • the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors). In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • some or all of the vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.
  • a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second vector comprising a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR;
  • the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived.
  • the immune cell is an aP T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.
  • a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR and ii) a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b)
  • the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived.
  • the immune cell is an aP T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.
  • nucleic acid sequences are swapped are also contemplated, such as where the first or second anti- GPC3 caTCR nucleic acid sequence is swapped with the anti-GPC3 CSR nucleic acid sequence or the c-Jun nucleic acid sequence.
  • a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR and ii) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, wherein the first and second anti-GPC3 caTCR nucleic acid sequences are under
  • the first promoter is operably linked to the 5’ end of the first anti-GPC3 caTCR nucleic acid sequence, and there is nucleic acid linker selected from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3’ end of first anti-GPC3 caTCR nucleic acid sequence to the 5’ end of the second anti-GPC3 caTCR nucleic acid sequence, wherein the first anti-GPC3 caTCR nucleic acid sequence and the second anti-GPC3 caTCR nucleic acid sequence are transcribed as a single RNA under the control of the promoter.
  • IRS internal ribosomal entry site
  • the first promoter is operably linked to the 5’ end of the second anti-GPC3 caTCR nucleic acid sequence, and there is nucleic acid linker selected from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3’ end of second anti-GPC3 caTCR nucleic acid sequence to the 5’ end of the first anti-GPC3 caTCR nucleic acid sequence, wherein the first anti-GPC3 caTCR nucleic acid sequence and the second anti-GPC3 caTCR nucleic acid sequence are transcribed as a single RNA under the control of the promoter.
  • IRS internal ribosomal entry site
  • the first and/or second promoters have the same sequence. In some embodiments, the first and/or second promoters have different sequences. In some embodiments, the first and/or second promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived.
  • the immune cell is an aP T cell and the TCR- TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of the immune cell.
  • nucleic acid sequences are swapped are also contemplated, such as where the first or second anti-GPC3 caTCR nucleic acid sequence is swapped with the anti-GPC3 CSR nucleic acid sequence or the c-Jun nucleic acid sequence.
  • a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a vector comprising a) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; c) an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR
  • the promoter is operably linked to one of the nucleic acid sequences, which is linked to the other nucleic acid sequences by nucleic acid linkers selected, individually, from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A), such that the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are transcribed as a single RNA under the control of the promoter.
  • the promoter is inducible.
  • the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived.
  • the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR- TMs of the introduced caTCR comprise sequences derived from TCR a and P chains.
  • the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits.
  • the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
  • the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of the immune cell.
  • the source of the engineered immune cells of the present disclosure may be a patient to be treated (z.e., autologous cells) or from a donor who is not the patient to be treated (e.g., allogeneic cells).
  • the engineered immune cells are engineered T cells.
  • the engineered T cells herein may be CD4 + CD8‘ (z.e., CD4 single positive) T cells, CD4'CD8 + (z.e., CD8 single positive) T cells, or CD4 + CD8 + (double positive) T cells.
  • the T cells may be cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells, or a mixture thereof.
  • the T cells to be engineered may be autologous or allogeneic.
  • T cells Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from a subject.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solutions with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (z.e., 3*28)- conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In some embodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this invention. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD- 14, CD20, CD1 lb, CD-16, HLA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 + , CD25 + , CD62Lhi, GITR + , and FoxP3 + .
  • T regulatory cells are depleted by anti-CD25 conjugated beads or other similar methods of selection.
  • the concentration of cells and surface can be varied. In some embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in some embodiments, a concentration of about 2 billion cells/ml is used. In some embodiments, a concentration of about 1 billion cells/ml is used. In some embodiments, greater than about 100 million cells/ml is used. In some embodiments, a concentration of cells of about any of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells of about any of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In some embodiments, a concentration of about 125 or about 150 million cells/ml is used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8 + T cells that normally have weaker CD28 expression.
  • T cells are obtained from a patient directly following treatment.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.
  • the T cells of the invention 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-stimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated, such as by contact with an anti-CD3 antibody, or antigenbinding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc . 30(8):3975-3977, 1998; Haanen et aL, J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(1- 2):53-63, 1999).
  • the cell culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • agents e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • the culture conditions include addition of IL-2, IL-7 and/or IL-15.
  • the cells to be engineered may be pluripotent or multipotent cells that are differentiated into mature T cells after engineering.
  • These non-T cells may be allogeneic and may be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem or progenitor cells.
  • pluripotent and multipotent cells are collectively called “progenitor cells” herein.
  • allogeneic cells are used, they are preferably engineered to reduce graft- versus-host rejection (e.g., by knocking out the endogenous B2M and/or TRAC genes).
  • cellular engineering of an immune cell comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., an anti-GPC3 caTCR and anti-GPC3 CSR), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell, function (e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression compared to a corresponding cell that has not been modified), or any combination thereof.
  • tumor specific targeting e.g., an anti-GPC3 caTCR and anti-GPC3 CSR
  • introduction of one or more endogenous genes that improve T cell function e.g., T cell
  • function e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression compared to
  • a cell can be engineered or modified with a transcription activator (e.g., CRISPR/Cas system-based transcription activator), wherein the transcription activator is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun).
  • a transcription activator e.g., CRISPR/Cas system-based transcription activator
  • the transcription activator is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun).
  • a cell described herein has been modified with a transcriptional activator, which is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun) in the cell.
  • a transcriptional activator refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to enhancers or promoter-proximal elements of a nucleic acid sequence and thereby inducing its transcription).
  • transcriptional activators that can be used with the present disclosure include: Transcription Activator-like Effector (TALE)-based transcriptional activator, zinc finger protein (ZFP)-based transcriptional activator, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system-based transcriptional activator, or a combination thereof.
  • TALE Transcription Activator-like Effector
  • ZFP zinc finger protein
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated protein
  • a cell described herein has been modified with a CRISPR/Cas- system-based transcriptional activator, such as CRISPR activation (CRISPRa).
  • CRISPR activation CRISPR activation
  • CRISPRa is a type of CRISPR tool that comprises the use of modified Cas proteins that lack endonuclease activity but retain the ability to bind to its guide RNA and the target DNA nucleic acid sequence.
  • modified Cas proteins which can be used with the present disclosure are known in the art (see, e.g., Pandelakis et al., Cell Systems (2020) 10(1): 1 - 14, which is incorporated herein by reference in its entirety).
  • the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as “dCas9”).
  • the modified Cas protein comprises a modified Casl2a protein.
  • a modified Cas protein that is useful for the present disclosure is bound to a guide polynucleotide (e.g., small guide RNA) (“modified Cas-guide complex”), wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun).
  • the guide polynucleotide comprises a recognition sequence that is complementary to the promoter region of an endogenous nucleic acid sequence encoding a protein of interest.
  • one or more transcriptional activators are attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when the modified Cas-guide complex is introduced into a cell, the one or more transcription activators can bind to a regulatory element (e.g., a promoter region) of an endogenous gene and thereby induce and/or increase the expression of the encoded protein (e.g., c-Jun).
  • a regulatory element e.g., a promoter region
  • c-Jun e.g., c-Jun
  • a regulatory element e.g., a promoter region
  • c-Jun e.g., c-Jun
  • common general activators that can be used include the omega subunit of RNAP, VP16, VP64 and p65 (see, e.g., Kabadi and Gersbach, Methods (2014) 69(2): 188-97).
  • one or more transcriptional repressors can be attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when introduced into a cell, the one or more transcriptional repressors can repress or reduce the transcription of a gene, e.g., such as those that can interfere with the expression of c-Jun (e.g., Bach2).
  • a modified Cas protein useful for the present disclosure can be attached to both one or more transcriptional activators and one or more transcriptional repressors.
  • a cell e.g., T cells
  • a recombinant antigen receptor e.g., an anti-GPC3 caTCR and an anti-GPC3 CSR
  • a protease e.g., tobacco etch virus (TEV)
  • sgRNA single guide RNA
  • the cell is modified to further comprise a linker for activation of T cells (LAT), complexed to the modified Cas protein attached to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., TEV-cleavable linker).
  • LAT T cells
  • a transcriptional activator e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)
  • VPR transcriptional activator
  • TEV-cleavable linker e.g., TEV-cleavable linker
  • a cell described herein has been modified using a combination of approaches.
  • a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding one or more proteins (e.g, an anti-GPC3 caTCR, an anti-GPC3 CSR, and a truncated EGFR (EGFRt)) and (ii) an exogenous transcriptional activator (e.g, CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun).
  • an exogenous nucleotide sequence encoding one or more proteins
  • an exogenous transcriptional activator e.g, CRISPRa
  • a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding a first protein (e.g., an anti-GPC3 caTCR), (ii) an exogenous nucleotide sequence encoding a second protein (e.g., an anti- GPC3 CSR), and (iii) an exogenous nucleotide sequence encoding a protein (e.g., a c-Jun protein).
  • the modified cell can further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt).
  • the exogenous nucleotide sequences encoding the first, second, and third proteins can be part of a single polycistronic vector.
  • the one or more exogenous nucleotide sequences and/or transcriptional activators can be introduced into a cell using any suitable methods known in the art.
  • suitable methods for delivering one or more exogenous nucleotide sequences to a cell include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.
  • a cell has been modified with a transcriptional activator (e.g., CRISPR/Cas-system-based transcription activator, e.g., CRISPRa), such that the expression of the endogenous c-Jun protein is increased compared to a corresponding cell that has not been modified with the transcriptional activator.
  • a transcriptional activator e.g., CRISPR/Cas-system-based transcription activator, e.g., CRISPRa
  • c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa).
  • CRISPRa a transcriptional activator
  • exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa).
  • transcriptional activator e.g., CRISPRa
  • the immune cells e.g., T cells
  • progenitor cells herein may be engineered to express an anti-GPC3 caTCR and an anti-GPC3 CSR, and overexpress c-Jun (e.g., a human c-Jun polypeptide).
  • the anti-GPC3 caTCR may specifically bind to GPC3 tumor cell
  • the anti-GPC3 CSR may bind specifically GPC3 (e.g., the same GPC3 but a different GPC3 epitope) on the tumor cell.
  • a receptor e.g., an anti-GPC3 caTCR or an anti- GPC3 CSR
  • a receptor is said to specifically bind to GPC3 when the KD for the binding is ⁇ 100 nM, e.g., ⁇ 10 nM or ⁇ 1 nM.
  • a KD binding affinity constant can be measured, e.g., by surface plasmon resonance (using, e.g., a BiacoreTM or OctetTM system).
  • the immune cell expresses the anti-GPC3 caTCR with even cell surface distribution.
  • the immune cell expresses the anti-GPC3 CSR with even cell surface distribution.
  • Even cell surface distribution can be characterized, for example, by staining patterns with continuous appearance and even thickness or signal intensity.
  • a composition such as a pharmaceutical composition, comprising anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun immune cells comprises fewer than about 10 % (such as fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) cells with aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface.
  • the caTCR+CSR+c-Jun T cell expresses the anti-GPC3 caTCR and/or anti-GPC3 CSR with less than about 10 % (such as less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface.
  • the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell expresses the anti-GPC3 caTCR and/or anti-GPC3 CSR with less than about 10 % (such as less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface.
  • the anti-GPC3 caTCR plus anti- GPC3 CSR plus human c-Jun T cell has a low level of antigen-independent anti-GPC3 caTCR and/or anti-GPC3 CSR activation.
  • the anti-GPC3 caTCR T cell has a low level of antigen-independent anti-GPC3 caTCR and/or anti-GPC3 CSR activation.
  • the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c- Jun T cell has a low level of T cell exhaustion.
  • T cell exhaustion naturally occurs during conditions of extended immune activation, such as with cancer or chronic infection, where T cells become dysfunctional. T cell exhaustion may be characterized by impaired effector function, prolonged expression of inhibitory receptors, and/or an altered transcriptional state compared to functional effector or memory T cells. Optimal clearance of tumor cells and infections is prevented by T cell exhaustion.
  • T cell exhaustion of the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell can be characterized by any means known in the art, for example, by determining its functional and/or phenotypic profile (Wherry, E. J., Nature immunology 12(6): 492-499, 2011; Jiang, Y., et al., Cell death & disease 6(6): el792, 2015).
  • the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell expresses low levels of one or more markers of T cell exhaustion, including, for example, PD-1, LAG-3, TIM-3, CTLA-4, and BTLA.
  • anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell maintains levels characteristic of nonexhausted T cells for IL-2 production, TNF-a production, IFN-y production, and granzyme B production, and/or maintains ex vivo killing capacity in the presence of target cells, suggesting that caTCR+CSR+c-Jun T cell is not undergoing self-activation and premature exhaustion.
  • anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun (“caTCR+CSR+c-Jun”) compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising an immune cell (such as a T cell) presenting on its surface an anti-GPC3 caTCR according to any of the anti-GPC3 caTCRs described herein, an anti- GPC3 CSR according to any of the CSRs described herein, and a c-Jun polypeptide.
  • the caTCR+CSR+c-Jun immune cell composition is a pharmaceutical composition.
  • the composition may comprise a homogenous cell population comprising caTCR+CSR+c-Jun immune cells of the same cell type and expressing the same anti-GPC3 caTCR, anti-GPC3 CSR, and c-Jun, or a heterogeneous cell population comprising a plurality of caTCR+CSR+c-Jun immune cell populations comprising caTCR+CSR+c-Jun immune cells of different cell types, expressing different anti-GPC3 caTCRs, expressing different anti-GPC3 CSRs, and/or expressing different c-Jun polypeptides (e.g., wildtype or mutant c- Jun polypeptides).
  • the composition may further comprise cells that are not caTCR+CSR+c- Jun immune cells.
  • cryopreserved/cryopreserving can be used interchangeably. Freezing includes freeze drying.
  • cryoprotective agents include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183: 1394- 1395; Ashwood- Smith, 1961 , Nature 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y. Acad.
  • DMSO can be used. Addition of plasma (e.g., to a concentration of 20-25%) can augment the protective effects of DMSO. After addition of DMSO, cells can be kept at 0° C until freezing, because DMSO concentrations of 1% can be toxic at temperatures above 4° C.
  • DMSO-treated cells can be pre-cooled on ice and transferred to a tray containing chilled methanol which is placed, in turn, in a mechanical refrigerator (e.g., Harris or Revco) at -80° C.
  • a mechanical refrigerator e.g., Harris or Revco
  • Thermocouple measurements of the methanol bath and the samples indicate a cooling rate of 1° to 3°C/minute can be preferred.
  • the specimens can have reached a temperature of - 80° C and can be placed directly into liquid nitrogen (-196° C).
  • the cells can be rapidly transferred to a long-term cryogenic storage vessel.
  • samples can be cryogenically stored in liquid nitrogen (-196° C) or vapor (-1° C). Such storage is facilitated by the availability of highly efficient liquid nitrogen refrigerators.
  • frozen cells can be thawed for use in accordance with methods known to those of ordinary skill in the art. Frozen cells are preferably thawed quickly and chilled immediately upon thawing.
  • the vial containing the frozen cells can be immersed up to its neck in a warm water bath; gentle rotation will ensure mixing of the cell suspension as it thaws and increase heat transfer from the warm water to the internal ice mass. As soon as the ice has completely melted, the vial can be immediately placed on ice.
  • methods can be used to prevent cellular clumping during thawing.
  • Exemplary methods include: the addition before and/or after freezing of DNase (Spitzer et al., 1980, Cancer 45:3075-3085), low molecular weight dextran and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20: 17-24), etc.
  • DNase Spitzer et al., 1980, Cancer 45:3075-3085
  • low molecular weight dextran and citrate low molecular weight dextran and citrate
  • hydroxyethyl starch Stiff et al., 1983, Cryobiology 20: 17-24
  • DMSO has no serious toxicity.
  • cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically-effective amount.
  • exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.
  • carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum.
  • HAS human serum albumin
  • a carrier for infusion includes buffered saline with 5% HAS or dextrose.
  • Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
  • Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • buffering agents such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls.
  • Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2- phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycol
  • compositions can include a local anesthetic such as lidocaine to ease pain at a site of injection.
  • a local anesthetic such as lidocaine to ease pain at a site of injection.
  • Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3 -pentanol.
  • Therapeutically effective amounts of cells within compositions can be greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than IO 10 cells, or greater than 10 11 cells.
  • cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater than 10 4 cells/ml, 10 7 cells/ml or 10 8 cells/ml.
  • nucleic acid compositions such as pharmaceutical compositions, also referred to herein as formulations
  • nucleic acid compositions comprising any of the nucleic acids encoding the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun polypeptides described herein.
  • the nucleic acid composition is a pharmaceutical composition.
  • the nucleic acid composition further comprises any of an isotonizing agent, an excipient, a diluent, a thickener, a stabilizer, a buffer, and/or a preservative; and/or an aqueous vehicle, such as purified water, an aqueous sugar solution, a buffer solution, physiological saline, an aqueous polymer solution, or RNase free water.
  • an aqueous vehicle such as purified water, an aqueous sugar solution, a buffer solution, physiological saline, an aqueous polymer solution, or RNase free water.
  • the amounts of such additives and aqueous vehicles to be added can be suitably selected according to the form of use of the nucleic acid composition.
  • compositions and formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage.
  • the compositions and formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection.
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
  • engineered immune cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically effective amount.
  • exemplary carriers include saline, buffered saline (e.g., phosphate buffered saline), physiological saline, water, Hanks' solution, Ringer’s solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. It is preferred that the carrier is isotonic.
  • the carrier can be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose.
  • HSA human serum albumin
  • Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol may also be included.
  • the pharmaceutical immune cell compositions may be administered in a therapeutically effective amount to a cancer patient systemically (e.g., through intravenous or portal vein injection) or locally (e.g., through intratumoral injection).
  • the compositions such as those targeting GPC3 are used to treat a patient with hepatocellular carcinoma (HCC), stomach cancer, pancreatic cancer, or a cancer in the reproductive system (see, e.g., Wang and Wang, Canadian J Gastroent Hep. (2016) art. 9049252).
  • HCC hepatocellular carcinoma
  • stomach cancer pancreatic cancer
  • a cancer in the reproductive system see, e.g., Wang and Wang, Canadian J Gastroent Hep. (2018) art. 9049252
  • treatment or “treating” refers to an approach for obtaining beneficial or desired results in the treated subject.
  • Such results include, but are not limited to: alleviating one or more symptoms resulting from the disease (e.g., HCC), diminishing the extent of the disease (e.g, reducing tumor volumes), stabilizing the disease (e.g, preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence or relapse of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, improving the quality of life, restoring body weight, and/or extension of survival (e.g., overall survival or progression-free survival).
  • HCC disease e.g., HCC
  • diminishing the extent of the disease e.g, reducing tumor volumes
  • stabilizing the disease e.g, preventing or delaying the worsening of the disease
  • preventing or delaying the spread e.g., metastasis
  • a therapeutically effective amount of the composition refers to the number of engineered immune cells (such as T cells) sufficient to achieve a desired clinical endpoint. In some embodiments, a therapeutically effective amount contains more than 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 of the engineered cells.
  • the pharmaceutical composition in some embodiments comprises the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful and can be determined.
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells
  • the cells and compositions in some embodiments are administered using standard administration techniques, formulations, and/or devices.
  • formulations and devices such as syringes and vials, for storage and administration of the compositions.
  • Administration can be autologous or allogeneic.
  • immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
  • Peripheral blood derived immunoresponsive cells or their progeny e.g., in vivo, ex vivo or in vitro derived
  • a therapeutic composition of the present disclosure e.g., a pharmaceutical composition containing a genetically modified cell
  • it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • the present disclosure provides pharmaceutical compositions comprising the nucleic acid molecules for expressing the anti-GPC3 caTCR, the anti-GPC3 CSR, and c-Jun.
  • the nucleic acid molecules may be as described above, such as the viral vectors (e.g., lentiviral vectors) described above.
  • the pharmaceutical compositions may be used ex vivo to engineer T or progenitor cells, which are then introduced to the patient.
  • compositions comprise the nucleic acid molecules or the recombinant viruses whose genome comprises the expression cassettes for the anti-GPC3 caTCR, the anti-GPC3 CSR, and c-Jun and a pharmaceutically acceptable carrier such as a buffered solution that optionally comprises other agents such as preservatives, stabilizing agents, and the like.
  • compositions may be provided as articles of manufacture, such as kits, that include vials (e.g., single-dose vials) comprising the biological materials (the cells or the nucleic acid molecules or recombinant viruses) and optionally instructions for use.
  • vials e.g., single-dose vials
  • biological materials the cells or the nucleic acid molecules or recombinant viruses
  • the expression constructs and/or compositions (such as pharmaceutical compositions) of the invention can be administered to individuals (e.g., mammals such as humans) to treat a disease and/or disorder involving abnormally high GPC3 expression (also referred to herein as a “GPC3 -positive” disease or disorder), including, for example, cancer (such as hepatocellular carcinoma (HCC)).
  • individuals e.g., mammals such as humans
  • GPC3 -positive disease or disorder also referred to herein as a “GPC3 -positive” disease or disorder
  • cancer such as hepatocellular carcinoma (HCC)
  • the present application provides a method of treating a GPC3-positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide.
  • a composition such as a pharmaceutical composition
  • the anti-GPC3 caTCR is any of the anti- GPC3 caTCRs provided herein.
  • the anti-GPC3 CSR is any of the anti- GPC3 CSRs provided herein.
  • the c-Jun polypeptide is any of the c-Jun polypeptides described herein.
  • a method of treating a cancer in an individual in need thereof comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and c-Jun polypeptides described herein.
  • a composition such as a pharmaceutical composition
  • a composition comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and c-Jun polypeptides described herein.
  • the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • the cancer is HCC.
  • the cancer is HCC and the treating comprises preventing the spread of the cancer, e.g., inhibiting (such as preventing) metastasis of the cancer.
  • the cancer is metastatic HCC.
  • the individual is human.
  • a method of treating a GPC3 -positive disease in an individual in need thereof comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligandbinding module that is capable of binding or interacting with GPC3; ii)
  • the TCRM of the anti-GPC3 caTCR is derived from a human y/8 TCR.
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30).
  • the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from CD30 (e.g., human CD30).
  • the human c- Jun polypeptide is a wildtype human c-Jun polypeptide.
  • the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • a method of treating a GPC3 -positive disease in an individual in need thereof comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module comprising: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC- CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and ii
  • the antigen binding module of the anti-GPC3 caTCR comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31.
  • the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5.
  • the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR.
  • the ligand binding module of the anti-GPC3 CSR comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 33.
  • the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30).
  • the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from CD30 (e.g., human CD30).
  • the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 44.
  • the anti- GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16.
  • the human c-Jun polypeptide is wildtype human c-Jun.
  • the human c- Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • HCC HCC
  • melanoma lung cancer
  • non-small cell lung cancer pancreatic cancer
  • breast cancer triple negative breast cancer
  • lung squamous cell carcinoma ovarian carcinoma
  • yolk sac tumor choriocarcinoma
  • neuroblastoma neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor testicular nonseminomatous germ cell tumor
  • gastric carcinoma gastric carcinoma
  • liposarcoma liposarcoma
  • a method of treating a GPC3 -positive disease in an individual in need thereof comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module comprising a scFv that is capable of binding or interacting with GPC3
  • a composition such as a pharmaceutical composition
  • the Fab comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 29.
  • the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 33.
  • the anti-GPC3 CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 44.
  • the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
  • the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • HCC HCC
  • melanoma lung cancer
  • non-small cell lung cancer pancreatic cancer
  • breast cancer triple negative breast cancer
  • lung squamous cell carcinoma ovarian carcinoma
  • yolk sac tumor choriocarcinoma
  • neuroblastoma neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor testicular nonseminomatous germ cell tumor
  • gastric carcinoma gastric carcinoma
  • liposarcoma liposarcoma
  • a method of treating a GPC3 -positive disease in an individual in need thereof comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing a polycistronic expression construct comprising an expression cassette for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide.
  • a composition such as a pharmaceutical composition
  • TCRM TCR module
  • the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33.
  • coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES).
  • the expression cassette comprises a constitutive or inducible promoter.
  • the promoter is an EF-la promoter.
  • the construct is a viral vector (such as a lentiviral vector).
  • the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
  • HCC HCC
  • melanoma lung cancer
  • non-small cell lung cancer pancreatic cancer
  • breast cancer triple negative breast cancer
  • lung squamous cell carcinoma ovarian carcinoma
  • yolk sac tumor choriocarcinoma
  • neuroblastoma neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor testicular nonseminomatous germ cell tumor
  • gastric carcinoma gastric carcinoma
  • liposarcoma liposarcoma
  • HepG2-luc Caliper, derived from ATCC HB-8065
  • Hep3B ATCC HB-8064
  • Both HepG2 and Hep3B are hepatocellular carcinoma (HCC) cell lines that express glypican-3 (GPC3).
  • the cell lines were cultured in RPMI-1640 (Gibco) or DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C/5% CO2.
  • GPC3-caTCR + GPC3-CD30-CSR contained a tri-cistronic expression cassette encoding the GPC3-specific caTCR (SEQ ID NO: 30 and 31) and the GPC3-specific CSR comprising a co-stimulatory domain derived from CD30, i.e., GPC3-CD30-CSR (SEQ ID NO: 16).
  • the second vector contained a quad-cistronic expression cassette encoding the GPC3- specific caTCR (SEQ ID NO: 30 and 31), the GPC3-CD30-CSR (SEQ ID NO: 16), and human c-Jun (SEQ ID NO: 1).
  • the expression cassette was under the transcriptional control of an EF- la promoter and the coding sequences for the different polypeptides were linked in frame by coding sequences for self-cleaving peptides (F2A, T2A or P2A, as illustrated in FIG. 1A).
  • the recombinant viruses were produced by transfection of 293T cells with vectors encoding the constructs using known lentiviral production protocols and packaging systems.
  • Example 1 Characterization of anti-GPC3 caTCR + anti-GPC3 CSR T cells overexpressing c-Jun in in vitro long-term target engagement assays
  • This Example describes the effects of c-Jun overexpression on the cytotoxicity of human T cells that were engineered to express an anti-GPC3 caTCR and an anti-GPC3 CSR comprising a co-stimulatory domain derived from CD30 (z.e., GPC3-CD30-CSR).
  • this Example shows that c-Jun overexpression promotes long-term target-cell- killing capabilities in T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR.
  • CD3+ T cells were prepared from PBMC-enriched whole blood using an EasySepTM Human T Cell Isolation Kit (StemCell Technologies), which negatively depletes cells expressing CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A.
  • Human T cells were activated and expanded with CD3/CD28 Dynabeads® (Invitrogen) according to the manufacturer’s protocol.
  • Activated T cells were cultured and maintained in RPMI- 1640 medium with 10% FBS plus 100 U/mL IL-2 and used at day 7-14.
  • the effector cells were normalized by caTCR expression (caTCR + ) based on human F(ab’)2 staining (adjusting all effector cell samples to the same percentage of receptor + (caTCR + ) cells among the total number of T cells using un-transduced/mock-transduced T cells).
  • T cells and target cells were co-cultured with starting effector-to-target ratio (E:T ratio) of 1 : 1.
  • E:T ratio effector-to-target ratio
  • 50,000 caTCR + T cells and 50,000 HepG2 cells were incubated together in each well in RPML1640 and 10% FBS with no cytokines.
  • the cells were re-challenged with 100,000 HepG2 cells per well every 7 days. The numbers of remaining target cells and caTCR + T cells were quantified after each target cell engagement.
  • Naive T cells are characterized by CCR7 + and CD45RA + , whereas memory T cells are characterized by CCR7 + , and CD45RA".
  • Table 3 the expression levels of CCR7 and the number of memory and naive T cells are comparable between T cells expressing Construct 1 and those expressing Construct 2.
  • CD3 + T cells were prepared and activated with CD3/CD28 Dynabeads® as described above.
  • the T cells were transduced with lentiviral Construct 1 or Construct 2 for 7-9 days.
  • the effector cells were normalized by caTCR + based on human F(ab’)2 staining.
  • the T cells were incubated and rechallenged with HepG2 cells, as described above.
  • PD-1, LAG-3, and TIM-3 are inhibitory receptors that accumulate on T cells as T cells lose function; thus, these molecules are used as markers for T cell exhaustion.
  • Example 2 In vivo efficacy of anti-GPC3 caTCR + anti-GPC3 CSR T cells overexpressing c-Jun in HepG2-luc mouse model re-challenge study
  • This Example shows that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR with c-Jun overexpression increased tumor-killing in vivo compared with T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.
  • mice in all treatment groups were subjected to tumor re-challenge studies, wherein HepG2-luc cells were implanted s.c. in the left flank around day 70 post T cell dosing.
  • T cells expressing Construct 2 performed significantly better than T cells expressing Construct 1, indicating c-Jun boosted the tumor-killing potential of the “GPC3-caTCR + GPC3-CD30-CSR” T cells in the tumor re-challenge study (FIG. 3). Additionally, in one study, mice injected with the higher dose of T cells expressing Construct 2 had higher peripheral blood T cell count (FIGs. 4A and 4B).
  • Example 3 In vivo efficacy of anti-GPC3 + anti-GPC3 CSR T cells overexpressing c- Jun in Hep3B mouse model
  • This Example shows that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR with c-Jun overexpression have increased T cell expansion in vivo compared with T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.
  • T cells expressing Construct 2 repressed the size of relapsing tumors to a higher extent than those expressing Construct 1.
  • the difference between the tumor repressing effects of Construct 1 and 2 as measured by tumor volume was less discernable.
  • mice in the Construct 2 groups in both doses had higher numbers of peripheral blood T cells, indicating c-Jun positively affects anti-GPC3 caTCR + anti-GPC3 CSR + T cell expansion in vivo (FIG. 4C)
  • Example 4 Characterization of T cells expressing various anti-GPC3 caTCRs + anti- GPC3 CSRs in in vitro long-term target engagement assays
  • This Example describes the cytotoxicity of human T cells that are engineered to express various anti-GPC3 caTCRs (e.g., GPC3-caTCR) comprising different sequences, and various anti-GPC3 CSRs comprising different sequences and a co-stimulatory domain derived from human CD30 (z.e., GPC3-CD30-CSR), in addition to a human c-Jun polypeptide.
  • various anti-GPC3 caTCRs e.g., GPC3-caTCR
  • various anti-GPC3 CSRs comprising different sequences and a co-stimulatory domain derived from human CD30 (z.e., GPC3-CD30-CSR)
  • T cells expressing each group of expression constructs are co-cultured with HepG2 cells, and assayed for cytotoxicity and exhaustion, as described in Example 1 above.
  • Example 5 Characterization of T cells expressing anti-GPC3 caTCRwith different anti-GPC3 CSRs
  • This Example characterizes human T cells that are engineered to express an anti- GPC3 caTCR (e.g, GPC3-caTCR) in combination with various anti-GPC3 CSRs (e.g., GPC3-CSR) comprising different co-stimulatory fragments (e.g., co-stimulatory fragments comprising a co-stimulatory domain and a transmembrane domain), in addition to a human c- Jun polypeptide.
  • GPC3 caTCR e.g, GPC3-caTCR
  • various anti-GPC3 CSRs e.g., GPC3-CSR
  • co-stimulatory fragments e.g., co-stimulatory fragments comprising a co-stimulatory domain and a transmembrane domain
  • Each exemplary GPC3 -caTCR encodes the VH/VL sequences of GPC3-3 (see, Sequence listing), and each exemplary GPC3- CSR encodes the VH/VL sequences of GPC3-2 (see, Sequence listing). Table 5.
  • Examples 5A-5G show that c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM- CD30 IC-CSR and c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR perform equally well, and better than the other anti-GPC3-CSRs.
  • T cells expressing each of the constructs described in Table 5 are co-cultured with HepG2 cells and tumor cytotoxicity was assayed by a Cytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH) released into culture supernatants, as described in Example 1.
  • Cytox 96® Non-Radioactive Cytotoxicity Assay Promega
  • T cells expressing either c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM-CD30 IC-CSR or c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR have higher killing efficacies than the other constructs outlined in Table 5, without a CSR or CSR comprising other costimulatory fragments.
  • the short-term killing ability of the various T cells expressing the constructs of Table 5 is also determined by measuring the amounts/levels of cytokines released from T cells upon engagement with HepG2 cells.
  • the levels of cytokine release in the supernatant after 16 hour co-culture are quantified with Luminex Magpix technology using BioRad Bio- Plex kits or by ELISA.
  • T cells with high cytotoxic potency secrete high levels of cytokines that are related to T cell activity, such as TNFa, GM-CSF, IFNy, and IL-2.
  • T cells expressing a c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have higher killing efficacies than those without any CSR, and higher than or about the same killing efficacies as those with CSRs that do not comprise a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains (see, Table 5).
  • T cells expressing each of the constructs of Table 5 are labeled with the intracellular dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and the dilution of the dye is observed as the T cells divide when stimulated with tumor cells. Persistence of the T cells is measured by counting the number of CFSE-positive cells remaining at an indicated day.
  • CFSE 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester
  • T cells expressing each of the constructs shown in Table 5 are serum starved overnight and labeled with CFSE using CellTrace CFSE (Thermo Fisher). 50,000 to 100,000 T cells are incubated at an effector cell to target cell ratio (E:T ratio) of 2: 1, and flow cytometry is used to observe serial dilution of the CFSE dye as the T cells divide at an indicated day. The total number of T cells are counted with FACS.
  • E:T ratio effector cell to target cell ratio
  • T cells expressing a c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain proliferate more than corresponding CAR T cells without any CSR, and proliferate more than or about the same as corresponding CAR T cells expressing CSRs that do not comprise a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
  • Example 5C In vitro T cell and tumor cell counts after multi-week engagements [0284] A FACS based assay for counting target cells is used to compare the long-term killing potential of T cells expressing each of the constructs described in Table 5. T cells expressing each of the constructs described in Table 5 show comparable survival post target cell engagement.
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain persist for a longer period of time over multiple engagements of tumor cells and kill more tumor cells than corresponding CAR T cells without CSR, and about the same as CAR T cells with CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains, if not better.
  • IC domain e.g., CD28 or 4- IBB IC domains
  • a FACS based assay for counting T cells and target cells is used to compare the long-term survival and target-cell killing potential of T cells expressing the constructs described in Table 5.
  • the T cells are co-cultured and re-challenged with target cells as described in Example 1.
  • the numbers of remaining target cells and total T cells are quantified with FACS on various days after each target cell engagement.
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain persist/survive for a longer period of time over multiple engagements of tumor target cells and kill more tumor cells than corresponding T cells without any CSR, and survive better and/or kill more tumor cells than or about the same as corresponding T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains.
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have decreased long term exhaustion markers than corresponding T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
  • Example 5E In vivo cytokine release
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain secrete higher levels of cytokines that are related to T cell activity, such as TNFa, GM-CSF, IFNy, and IL-2, than corresponding T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
  • Example 5F Differentiation of T-cell subsets over time (CCR7/CD45RA) and memory T cell quantification
  • FACS analysis of T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain show a greater level of expression of the T cell differentiation markers CCR7 and CD45RA , as well as increased percentage of memory and naive T cells compared to T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains, prior to target engagement.
  • IC domain e.g., CD28 or 4- IBB IC domains
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3-CD30-CSR develop into and maintain a high memory T cell population after target stimulation, including central memory and effector memory T cells.
  • the cell surface expression of memory T cell markers CCR7 and CD45RA is measured.
  • T cells with high CCR7 expression levels and low CD45RA expression levels are considered central memory T cells
  • T cells with low CCR7 and low CD45RA expression levels are considered effector memory T cells
  • T cells with low CCR7 and high CD45RA expression levels are considered effector T cells
  • T cells with high CCR7 and high CD45RA are naive T cells which are the initial type of T cells before target/antigen challenge/recognition (Mahnke et al., Eur J Immunol. 43(11):2797-809, 2013).
  • naive T cells proliferate and differentiate into effector cells, most of which carry out the job of destroying targets and then die, while a small pool of T cells ultimately develops into long- lived memory T cells which can store the T cell immunity against the specific target.
  • the central memory T cells are found to have longer lives than effector memory T cells and are capable of generating effector memory T cells, but not vice versa. Therefore, the ability to develop into and maintain memory T cells, especially central memory T cells, is an important and desired feature for potentially successful T cell therapies.
  • T cells expressing c-Jun and an anti-GPC3 caTCR but no CSR are incubated with HepG2 cells at an E:T ratio of 2: 1 (e.g., 100,000 receptor + T cells and 50,000 HepG2 cells in each well on a 96- well plate) for 7 days. The cells are then re-challenged with 50,000- 100,000 HepG2 cells per well every 7 days.
  • the T cells in the other groups expressing different CSRs are incubated with target cells at an E:T ratio of 1 :2 (e.g., 25,000 receptor + T cells and 50,000 HepG2 cells in each well) for 7 days. The cells are then re-challenged with 50,000-100,000 HepG2 cells per well every 7 days.
  • the T cell and HepG2 cell mixtures are diluted 1 :6 before the fourth and fifth HepG2 cell engagement, to avoid the overcrowding of T cells due to the significant T cell expansion, so that only one sixth of the previously remaining cells are rechallenged with 50,000-100,000 HepG2 cells.
  • the entire cell mixture in a well from each sample is stained with antibodies against CCR7 and CD45RA and analyzed by flow cytometry.
  • Receptor + T cell numbers are counted, and cells are grouped into various T cell types based on their CCR7 and CD45RA expression levels: central memory T cells (CD45RA'CCR7 + ), effector memory T cells (CD45RA'CCR7'), effector T cells (CD45RA+ CCR7" ), and naive T cells (CD45RA + CCR7 + ). Percentages of various types of T cells among the total number of receptor + T cells are calculated. In some experiments, the cells are also stained with antibodies against CD8 or CD4 to determine the CD8 (cytotoxic T cells) and CD4 (helper T cells) composition of the T cell types.
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR are measured before and after target cell engagement.
  • T cells expressing c- Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain are able to develop into and maintain high numbers and high percentages of central memory T cells upon engagement with HepG2 cells, higher than those without any CSR or those expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
  • Example 5G In Vivo efficacy of T cells in a HepG2 or a Hep3B mouse model [0299] About 10 7 HepG2 tumor cells or Hep3B are implanted subcutaneously in NSG mice and allowed to form a solid tumor mass 150mm 3 . 5xl0 6 T cells expressing each of the constructs described in Table 5 are injected i.v. into the tumor bearing mice. 3 weeks after T- cell dosing, the mice are sacrificed and tumors removed, fixed, and sectioned onto slides. Tumor sections are stained with CD3 antibody to visualize the T cells that are present within the solid tumor. Quantification of the number of CD3 + cells can be used to score the tumor infiltration ability of the T cells (T-cell/mm 2 ).
  • T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have higher in vivo tumor infiltration/penetration rates/levels (z.e., higher numbers of T cells/mm 2 ) than those without any CSR or those expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains.

Abstract

The present disclosure provides compositions and methods for improving immune cell therapy. In particular aspects, the present disclosure relates to expression construct(s) expressing a chimeric antibody-T cell receptor (TCR) construct (caTCR) and a chimeric stimulating receptor (CSR) that both specifically target glypican 3 (GPC3), along with a human c-Jun polypeptide.

Description

IMMUNE CELL THERAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Application 63/369,171, filed July 22, 2022. The disclosure of the aforementioned provisional application is incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing is hereby incorporated by reference in its entirety. The XML copy, created on June 26, 2023 is named 026225. WO027.xml and is 109,508 bytes in size.
BACKGROUND OF THE INVENTION
[0003] In the past two decades, fundamental advances in immunology and tumor biology, combined with the identification of a large number of tumor antigens, have led to significant progress in the field of cell-based immunotherapy. T cell therapy, which aims to treat cancer by transferring autologous and ex vivo expanded T cells to patients, has resulted in some notable antitumor responses (Blattman et al., Science. (2004) 305(5681):200-5). For example, the administration of naturally occurring tumor infiltrating lymphocytes (TILs) expanded ex vivo mediated an objective response rate ranging from 50-70% in melanoma patients, including patients with bulky invasive tumors at multiple sites involving liver, lung, soft tissue and brain (Rosenberg et al., Nat Rev Cancer. (2008) 8(4):299-308; Dudley et al., J Clin Oncol. (2005) 23(10):2346-57).
[0004] A major limitation to the widespread application of TIL therapy is the difficulty in generating human T cells with antitumor potential. As an alternative approach, exogenous high-affinity TCRs can be introduced into normal autologous T cells of the patients through T cell engineering. The adoptive transfer of these cells into lympho-depleted patients has been shown to mediate tumor regression in cancers such as melanoma, colorectal carcinoma, and synovial sarcoma (Kunert et al., Front Immunol. (2013) 4:363; Robbins et al., Clin Cancer Res. (2015) 21(5): 1019-27). One of the advantages of TCR-engineered T cell therapy is that it can target the entire array of potential intracellular tumor-specific proteins, which are processed and delivered to the cell surface through MHC presentation; these antigens can be recognized even at a low density by antigen-specific cytotoxic T cells (Kunert, supra). [0005] Attempts have been made to engineer TCR molecules having antibody specificity with T cell receptor effector functions. In some of these approaches, the variable and constant domains of a TCR (e.g., an aP TCR or a y5 TCR) are replaced by the variable and constant domains of an antibody against a tumor antigen, creating a chimeric antibody-TCR called “abTCR” or “caTCR.” See, e.g, WO 2017/070608 and WO 2018/200582, the disclosures of which are incorporated by reference herein in their entirety. In one of the approaches, a chimeric stimulating receptor (CSR) is employed in combination with a caTCR to enhance the tumor-killing efficacy of the engineered T cells. Like a chimeric antigen receptor (CAR), a CSR has an extracellular domain that binds a target ligand, e.g., a tumor antigen, and an intracellular co-stimulatory domain, but unlike a CAR, the CSR does not have an intracellular primary immune cell signaling domain (which typically is a CD3 zeta chain’s intracellular domain) or a functional primary immune cell signaling domain. The CSR and the caTCR may bind to different epitopes of the same target/antigen and work synergistically to boost the cytotoxicity of the engineered T cells. See, e.g., WO 2018/200583, the disclosure of which is incorporated by reference herein in its entirety.
[0006] One challenge facing T cell therapy is the lack of persistence of T cells in vivo due to a phenomenon known as T cell exhaustion. See, e.g., Fraietta et al., Nat Med. (2018) 24(5):563-71; Long et al., Nat Med. (2015) 21(6):581-90; and Eyquem et al., Nature (2017) 543(7643): 113-7. T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., reduced cytokine secretion and cytotoxicity), expression of multiple surface inhibitory receptors, and apoptosis. T cell exhaustion has been attributed to constant antigen exposure, leading to continuous TCR signaling, or to tonic antigen-independent signaling through an engineered antigen receptor on T cells (see, e.g., Long, supra). Prevention or reversal of T cell exhaustion has been sought as a means to enhance T cell effectiveness, e.g., in patients with cancer or chronic infections and in T cell therapy. See, e.g., WO 2019/118902, the disclosure of which is incorporated by reference herein in its entirety.
[0007] Glypican 3 (GPC3, also known as SGB, DGSX, MXR7, SDYS, SGBS, OCL5, SGBS1, GTR2-2) is a cell surface protein that is overexpressed in multiple cancer types, including many solid tumors, such as hepatocellular carcinoma (HCC), melanoma (Nakatsura T et al., Clin Cancer Res. (2004) 10(19) : 6612-21 ), lung squamous cell carcinoma (Yu X et al., Genet Mol Res. (2015) 14(3): 10185-92), ovarian carcinoma (Stadlmann S et al, IntJ Gynecol Pathol. (2007) 26(3):341-4), yolk sac tumor, choriocarcinoma (Zynger DL et al., Am J Surg Pathol. (2006) 30(12): 1570-5), Wilms’ tumor, and liposarcoma (Baumhoer D et al., Am J Clin Pathol. (2008) 129(6):899-906). There remains a medical need for developing therapies against various cancers including those overexpressing GPC3.
[0008] Accordingly, there remains a need for improved T cell therapy in which the engineered T cells have high as well as sustained tumor-killing potency, and in particular, improved T cell therapy in which the T cells specifically target GPC3 -overexpressing cancers.
SUMMARY OF THE INVENTION
[0009] The present disclosure provides compositions and methods for improving immune cell therapy. In some aspects, provided herein are one or more expression constructs comprising one or more expression cassettes for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligand-binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide.
[0010] In some embodiments, the c-Jun is a wildtype human c-Jun. In some embodiments, the wildtype human c-Jun comprises at least about 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the c-Jun is a mutant human c-Jun. In some embodiments, mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain. In some embodiments, the c-Jun comprises: (i) S63 A and S73 A substitutions as compared to SEQ ID NO: 1; or, (ii) a deletion between amino acid residues 2 and 102 or between amino acid residues 30 and 50 as compared to SEQ ID NO: 1.
[0011] In some embodiments, the GPC3 is a cell-surface bound GPC3.
[0012] In some embodiments, the TCRM is derived from a human y/8 TCR. In some embodiments, the caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the caTCR. In some embodiments, the stabilization module is selected from the group consisting of a TCR constant domain, a CH1-CL module, a CH2-CH2 module, a CH3- CH3 module, and a CH4-CH4 module. In some embodiments, the stabilization module is derived from a human protein. In some embodiments, the CL contained in the CH1-CL module is derived from a kappa antibody light chain or lambda antibody light chain. In some embodiments, the antigen-binding module of the caTCR is a Fab, a Fab', a (Fab')2, an Fv, or a single chain Fv (scFv).
[0013] In some embodiments, the antigen-binding module of the caTCR comprises: (i) a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 (HC-CDR1) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable domain (VL) comprising a light chain complementarity determining region (LC-CDR) 1 (LC-CDR1) comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; or, (iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 3 amino acid substitutions. In some embodiments, the antigen-binding module of the caTCR comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC- CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC- CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or, (iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
[0014] In some embodiments, the antigen-binding module of the caTCR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 29. In some embodiments, the antigen-binding module of caTCR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
[0015] In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR 5 chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR y chain. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR y chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR 5 chain. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 5. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 63, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 64. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 65, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 66. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 67, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 68. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 69, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 70.
[0016] In some embodiments, the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30, CD28, CD3s, CD3< CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30. [0017] In some embodiments, the co-stimulatory immune cell signaling module is derived from the intracellular domain of a co-stimulatory receptor of a TCR. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD30, CD28, 4-IBB, 0X40, ICOS, CD27, and CD40. In some embodiments, the co-stimulatory immune cell signaling module of the CSR is derived from human CD30. In some embodiments, the co- stimulatory immune cell signaling module of human CD30 comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the co-stimulatory immune cell signaling module of human CD30 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the co-stimulatory immune cell signaling module and the transmembrane domain of the CSR are both derived from CD30 (e.g., human CD30).
[0018] In some embodiments, the ligand-binding module of the CSR comprises: (i) a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 (HC-CDR1) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable domain (VL) comprising a light chain complementarity determining region (LC-CDR) 1 (LC-CDR1) comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; or, (iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 3 amino acid substitutions. In some embodiments, the ligand-binding module of the CSR comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or, (iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC- CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
[0019] In some embodiments, the ligand-binding module of the CSR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 29. In some embodiments, the ligand-binding module of the CSR comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
[0020] In some embodiments, the ligand-binding module CSR comprises: (i) SEQ ID NO: 32, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 32; (ii) SEQ ID NO: 33, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 33; or, (iii) SEQ ID NO: 34, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 34.
[0021] In some embodiments, (i) the antigen-binding module of the caTCR comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and (ii) the ligand-binding module of the CSR comprises: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
[0022] In some embodiments, the antigen-binding module of the caTCR comprises a Fab that binds to GPC3, wherein the ligand-binding module of the CSR comprises a scFv that binds to GPC3, and wherein the transmembrane module and co-stimulatory immune cell signaling module of the CSR are both derived from CD30 (e.g., human CD30).
[0023] In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
[0024] In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34.
[0025] In some embodiments, the construct(s) are viral vectors. In some embodiments, the viral vector(s) are selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein- Barr viral vectors.
[0026] In some embodiments, the expression construct comprises a polycistronic expression cassette for expressing the caTCR, the CSR, and the c-Jun.
[0027] In some aspects, provided herein is a polycistronic expression construct comprising an expression cassette for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c- Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 34. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 26 and 27, and a coding sequence for SEQ ID NO: 34. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 32. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 24 and 25, and a coding sequence for SEQ ID NO: 34.
[0028] In some embodiments, the coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF-la promoter. In some embodiments, the construct is a viral vector. In some embodiments, the viral vector is selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors. In some embodiments, provided herein is a recombinant virus comprising the polycistronic expression construct of any of the preceding embodiments. In some embodiments, the polycistronic expression construct is a lentiviral vector. [0029] In some aspects, provided herein is a method of engineering immune cells, comprising: (a) providing a starting cell population, (b) introducing the expression construct(s) of any one of the preceding claims, or the recombinant virus of any one of the preceding claims into the starting cell population, (c) optionally selecting cells that express the caTCR, the CSR, and the c-Jun, and (d) deriving engineered immune cells from the cells of step (b) or (c). In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the starting cell population comprises immune cells. In some embodiments, the starting cell population comprises autologous or allogeneic T cells. In some embodiments, the starting cell population comprises pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells.
[0030] In some aspects, provided herein is a population of human cells comprising the expression construct(s) of any of the preceding embodiments or the recombinant virus of any of the preceding embodiments. In some embodiments, the human cells are immune cells. In some embodiments, the population of immune cells are obtained by the method of any of the preceding embodiments. In some embodiments, the immune cells are human cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune cells express a lower level of an exhaustion marker compared to corresponding cells that do not overexpress c-Jun. In some embodiments, the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3. [0031] In some aspects, provided herein is a pharmaceutical composition comprising the expression con struct! s) of any of the preceding embodiments, the recombinant virus of any of the preceding embodiments, or the cells of any of the preceding embodiments, and a pharmaceutically acceptable carrier.
[0032] In some aspects, provided herein is a method of killing target cells, comprising contacting the target cells with the pharmaceutical composition of any of the preceding embodiments under conditions that allow killing of the target cells by the immune cells, wherein the target cells express the GPC3. In some embodiments, the immune cells express a lower level of an exhaustion marker when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the target cells are cancer cells. In some embodiments, the co-stimulatory immune cell signaling module in the CSR is derived from human CD30 and the immune cells express a lower level of an exhaustion marker, as compared to corresponding immune cells engineered to express a CSR whose costimulatory immune cell signaling module is derived from human CD28. In some embodiments, the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.
[0033] In some aspects, provided herein is a method of treating an individual in need thereof, comprising administering the pharmaceutical composition of any of the preceding embodiments to the individual. In some aspects, provided herein is a use of the expression con struct! s) of any of the preceding embodiments, the recombinant virus of any of the preceding embodiments, or the pharmaceutical composition of any of the preceding embodiments, for the manufacture of a medicament for treating an individual in need thereof. In some aspects, provided herein is a use of the expression construct(s) of any of the preceding embodiments, the recombinant virus of any of the preceding embodiments, or the pharmaceutical composition of any of the preceding embodiments, for the treatment of an individual in need thereof. In some embodiments, the individual has a GPC3-positive disease. In some embodiments, the GPC3-positive disease is cancer. In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
[0034] In some aspects, provided herein is a method of reducing exhaustion of an engineered immune cell, comprising introducing to the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises the expression construct s) of any one of the preceding embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.
[0036] FIG. 1A is a schematic representation of an exemplary pair of caTCR and CSR (the top panel) and the two expression constructs used in in vivo efficacy studies of T cells described herein (the bottom panel). Construct 1 encodes an anti-GPC3 caTCR (“anti-GPC3- caTCR”) plus an anti-GPC3 CSR comprising a co-stimulatory domain derived from CD30 (“anti-GPC3-CD30-CSR”), whereas Construct 2 encodes “anti-GPC3-caTCR”+“anti-GPC3- CD30-CSR”, and a“c-Jun” polypeptide.
[0037] FIG. IB is a schematic representation of the design of in vivo efficacy studies of T cells expressing anti-GPC3 -caTCR and anti-GPC3-CD30-CSR, with or without c-Jun overexpression, in two different mouse models.
[0038] FIG. 2 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression, in HepG2-luc mouse model. The T cells were isolated from two healthy human donors. For each T cell type, two doses were tested (5* 106 and 2* 106).
[0039] FIG. 3 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression in HepG2-luc mouse model in tumor re-challenge studies, wherein the animals were implanted with tumor cells again after the initial tumors regressed.
[0040] FIGs. 4A-4C are graphs showing peripheral blood T cell counts in HepG2-luc and Hep3B mice injected with anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells with or without c-Jun overexpression. In FIG. 4A, the T cells were isolated from Donor A and the mouse model was HepG2-luc; in FIG 4B, the T cells were isolated from Donor B and the mouse model was HepG2-luc; in FIG. 4C, the T cells were isolated from Donor C and the mouse model was Hep3B.
[0041] FIG. 5 is a graph showing the in vivo tumor killing effects of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells, with or without c-Jun overexpression, in Hep3B mouse model.
DETAILED DESCRIPTION
[0042] The present application provides engineered human cells (e.g., immune cells such as T cells) comprising constructs for expressing a chimeric antibody-T cell receptor (caTCR), a chimeric stimulating receptor (CSR; also termed “chimeric signaling receptor” herein), and a c-Jun polypeptide (e.g., a human c-Jun polypeptide). Both the caTCR and the CSR specifically bind or interact with GPC3 (also referred to as “anti-GPC3 caTCR” and “anti- GPC3 CSR”, respectively, herein). The anti-GPC3 caTCR comprises an antigen-binding module that specifically binds to GPC3, and a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. The anti-GPC3 CSR comprises a ligand-binding domain that specifically binds to GPC3, and a co-stimulatory immune cell signaling domain capable of providing a stimulatory signal to the immune cell and does not comprise a functional primary T cell signaling sequence. The GPC3 targeted by the anti- GPC3 caTCRs and anti-GPC3 CSRs provided herein may be expressed on the cell surface of a target cell (e.g., a diseased cell). In some embodiments, the anti-GPC3 caTCR and the anti- GPC3 CSR bind to different regions on the same GPC3 protein. The disease may be a cancer such as a cancer of the gastrointestinal system, for example, gastric or liver cancer. The anti- GPC3 caTCR is regulated by the naturally occurring machinery that controls TCR activation, while the anti-GPC3 CSR potentiates the immune response mediated by the anti-GPC3 caTCR. c-Jun expression helps sustain the active state of the T cells by, e.g., alleviating or preventing T cell exhaustion.
[0043] The present engineered immune cells, such as T cells, exhibit sustained, potent cytotoxicity against target-bearing tumor cells. As compared to T cells that do not overexpress c-Jun (e.g., through an exogenously introduced nucleic acid sequence encoding c-Jun), the present engineered T cells display fewer signs of T cell exhaustion. The engineered cells may have one or more of the following characteristics: (i) they do not have increased expression of exhaustion markers PD-1, TIM-3 and/or LAG-3 over time, (ii) have reduced rates of apoptosis, (iii) they have increased memory cell formation and/or maintenance of memory markers (e.g., CCR7 and CD45RA); (iv) they have enhanced cytotoxicity; (v) they display increased recognition of tumor targets with low surface antigen; (vi) they have enhanced proliferation in response to antigen; (vii) maintain survival and functionality after repeated antigen stimulation; and (vii) they display increased tumorinfiltrating abilities.
[0044] In some embodiments, the anti-GPC3 CSR comprises a co-stimulatory immune cell signaling domain derived from the intracellular domain of CD30 (e.g., human CD30). The present inventors have unexpectedly discovered that c-Jun overexpression significantly reduces exhaustion in T cells engineered to express anti-GPC3 caTCR and a CD30-based anti-GPC3 CSR.
I. Definitions
[0045] The term “antibody” or "antibody moiety" includes full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions (ie., variable domains) of the light and heavy chains are responsible for antigen-binding. The variables region in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions ofKabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 5, a, y, and p heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (yl heavy chain), lgG2 (y2 heavy chain), lgG3 (y3 heavy chain), lgG4 (y4 heavy chain), IgAl (al heavy chain), or lgA2 (a2 heavy chain).
[0046] The term "antigen-binding fragment" as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
[0047] As use herein, the term "specifically binds" or "is specific for" refers to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody moiety that specifically binds to a target (which can be an epitope) is an antibody moiety that binds the target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets. In some embodiments, an antibody moiety that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (for example a cell surface antigen or a peptide/MHC protein complex) with a binding affinity that is at least about 10 times its binding affinity for other targets.
[0048] The term “T cell receptor,” or “TCR,” refers to a heterodimeric receptor composed of aP or y5 chains that pair on the surface of a T cell. Each a, P, y, and 5 chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR), followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunits of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523- 544; Murphy (2012), xix, 868 p.).
[0049] The term “TCR-associated signaling molecule” refers to a molecule having a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) that is part of the TCR- CD3 complex. TCR-associated signaling molecules include CD3ys, CD35s, and ( (also known as CD3(^ or CD3( ).
[0050] “Activation”, as used herein in relation to a cell expressing CD3, refers to the state of the cell that has been sufficiently stimulated to induce a detectable increase in downstream effector functions of the CD3 signaling pathway, including, without limitation, cellular proliferation and cytokine production.
[0051] The term “module” when referring to a portion of a protein is meant to include structurally and/or functionally related portions of one or more polypeptides which make up the protein. For example, a transmembrane module of a dimeric receptor may refer to the portions of each polypeptide chain of the receptor that span the membrane. A module may also refer to related portions of a single polypeptide chain. For example, a transmembrane module of a monomeric receptor may refer to portions of the single polypeptide chain of the receptor that span the membrane. A module may also include only a single portion of a polypeptide. [0052] The term “isolated nucleic acid” as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
[0053] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al, J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262'.'l32-'l 45 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al, Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Pliickthun, J. Mol. BioL, 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al, Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present invention and for possible inclusion in one or more claims herein. The specific CDR sequences of the present invention are defined by the IMGT numbering system. Table 1. CDR Definitions.
Kabat1 Chothia2 MacCallum3 IMGT4 AHo5
VH CDRI 31-35 26-32 30-35 27-38 25-40
VH CDR2 50-65 53-55 47-58 56-65 58-77
VH CDR3 95-102 96-101 93-101 105-117 109-137
VL CDRI 24-34 26-32 30-36 27-38 25-40
VL CDR2 50-56 50-52 46-55 56-65 58-77
VL CDR3 89-97 91-96 89-96 105-117 109-137
’ Residue numbering follows the nomenclature of Kabat et al., supra 2Residue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Pltickthun, supra
[0054] The term “chimeric antibodies” refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
[0055] The term “semi-synthetic” in reference to an antibody or antibody moiety means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.
[0056] The term “fully synthetic” in reference to an antibody or antibody moiety means that the antibody or antibody moiety has fixed naturally occurring VH/VL framework pairings, but non-naturally occurring (i.e., synthetic) sequences of all 6 CDRs of both heavy and light chains.
[0057] “Fv” is the minimum antibody fragment which contains a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[0058] “Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
[0059] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0060] “Homology” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are “homologous” at that position. The “percent of homology” or “percent sequence identity” between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100, considering any conservative substitutions as part of the sequence identity. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792- 1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004).
[0061] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
[0062] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
[0063] The term "inducible promoter" refers to a promoter whose activity can be regulated by adding or removing one or more specific signals. For example, an inducible promoter may activate transcription of an operably linked nucleic acid under a specific set of conditions, e.g., in the presence of an inducing agent or conditions that activates the promoter and/or relieves repression of the promoter.
[0064] As used herein, the term “cell engineering” or “cell modification” (including derivatives thereof) refers to the targeted modification of a cell, e.g., an immune cell disclosed herein. [0065] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of the disease (such as, for example, tumor volume in cancer). The methods of the invention contemplate any one or more of these aspects of treatment.
[0066] The terms “recurrence,” “relapse” or “relapsed” refers to the return of a cancer or disease after clinical assessment of the disappearance of disease. A diagnosis of distant metastasis or local recurrence can be considered a relapse.
[0067] The term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment.
[0068] An “effective amount” of a caTCR or composition comprising a caTCR as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and by known methods relating to the stated purpose. [0069] The term “therapeutically effective amount” refers to an amount of a caTCR or composition comprising a caTCR as disclosed herein, effective to “treat” a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of a caTCR or composition comprising a caTCR as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent a caTCR or composition comprising a caTCR as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient. In the case of infectious disease, such as viral infection, the therapeutically effective amount of a caTCR or composition comprising a caTCR as disclosed herein can reduce the number of cells infected by the pathogen; reduce the production or release of pathogen- derived antigens; inhibit (z.e., slow to some extent and preferably stop) spread of the pathogen to uninfected cells; and/or relieve to some extent one or more symptoms associated with the infection. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient.
[0070] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
[0071] It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of’ embodiments.
[0072] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".
[0073] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
[0074] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.
[0075] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of immunology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
II. Expression Constructs
[0076] The present invention provides one or more expression constructs comprising one or more expression cassettes for expressing a caTCR (e.g., a caTCR comprising an antigenbinding module that specifically binds to GPC3), a CSR (e.g., a CSR comprising a ligandbinding module that is capable of binding or interacting with GPC3), and a c-Jun polypeptide (e.g., a human c-Jun polypeptide), according to any of the caTCRs, CSRs, and c-Jun polypeptides described herein (such expression construct(s) is also referred to herein as a “caTCR+CSR+c-Jun expression construct s)”). The caTCRs and CSRs of the present invention specifically target GPC3 (such caTCRs and CSRs are also referred to herein as “anti-GPC3 caTCRs” and “anti-GPC3 CSRs”, respectively). It should be understood that when used to describe expression constructs or expression cassettes, the words "express/expressing" and "encode/encoding" are used interchangeably herein.
[0077] In some aspects, provided herein is one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligandbinding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the anti-GPC3 CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the expression con struct! s) are viral vectors. In some embodiments, the viral vector(s) are selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors. In some embodiments, the expression construct(s) express a polycistronic expression cassette for expressing the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun. In some embodiments, the TCRM of the anti-GPC3 caTCR is derived from a human y/8 TCR. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30). In some embodiments, the co-stimulatory immune cell signaling module of the anti-GPC3 CSR is derived from CD30 (e.g., human CD30). In some embodiments, the human c-Jun polypeptide is a wildtype human c-Jun polypeptide.
[0078] In some embodiments, provided herein one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module comprising: (1) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (2) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module comprising: (1) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (2) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO: 17; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a costimulatory signal to the immune cell, wherein the ligand-binding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3 caTCR comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti- GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the ligand binding module of the anti-GPC3 CSR comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30). In some embodiments, the co-stimulatory immune cell signaling module of the anti-GPC3 CSR is derived from CD30 (e.g., human CD30). In some embodiments, the human c-Jun polypeptide is wildtype human c-Jun. In some embodiments, the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1. [0079] In some embodiments, provided herein is one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module comprising a scFv that is capable of binding or interacting with GPC3; ii) a transmembrane module derived from CD30; and iii) a co-stimulatory immune cell signaling module derived from CD30 that is capable of providing a co-stimulatory signal to the immune cell, wherein the anti-GPC3 CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the Fab comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-GPC3 CSR comprises a fragment of CD30. In some embodiments, the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.
[0080] In other aspects, provided herein is a polycistronic expression construct comprising an expression cassette for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 16. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 16. In some embodiments, the expression cassette further comprises a coding sequence for a signal peptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 2. In some embodiments, coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF- la promoter. In some embodiments, the construct is a viral vector (such as a lentiviral vector).
A. Chimeric Antibody-T Cell Receptor (caTCR) Constructs
[0081] The expression constructs provided herein encodes a chimeric antibody-T cell receptor (caTCR) construct comprising an antigen binding module that specifically binds to GPC3 (also referred to herein as an “anti-GPC3 caTCR”). Exemplary caTCRs are provided in PCT/US2016/058305, the contents of which is incorporated herein by reference in its entirety. The anti-GPC3 caTCR can specifically bind to GPC3 and is capable of recruiting at least one TCR-associated signaling molecule (such as CD35s, CD3ys, and/or CQ. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that specifically binds to a cell surface-bound GPC3. In some embodiments, there is provided an anti-GPC3 caTCR comprising a) an antigen-binding module that specifically binds to GPC3, and b) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) derived from one of the transmembrane domains of a naturally occurring TCR (such as an aPTCR or a ySTCR) and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring TCR (such as an aPTCR or a ySTCR), wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule (such as CD36s, CD3ys, and/or i ), and wherein the antigen-binding module is linked to the first and/or second TCRDs. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. [0082] The anti-GPC3 caTCRs described herein may have one or more features described in this section. It is intended that any of the features for each component of the anti-GPC3 caTCR (e.g., antigen-binding module, TCRD, TCR-TM, spacer module, stabilization module, T cell co-stimulation sequences, various linkers, etc.) described herein can be combined with each other, with any of the features of the anti-GPC3 CSR, and with c-Jun as if each and every combination is individually described.
[0083] In some embodiments, the antigen-binding module (such as an antibody moiety) can specifically bind to GPC3 with a) an affinity that is at least about 10 (including for example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity for other molecules; or b) a Kd no more than about 1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its Kd for binding to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.
[0084] In some embodiments, the stabilization module comprises a fist stabilization domain and a second stabilization domain. In some embodiments, the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilization module is located between the antigenbinding module and the TCRM. In some embodiments, the stabilization module is derived from an antibody moiety. In some embodiments, the stabilization module is derived from an antibody constant domain. In some embodiments, the stabilization module is derived from an antibody heavy chain constant domain. In some embodiments, antibody heavy chain constant domains contained in the stabilization module are derived from an IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgA (c.g, IgAl or IgA2), IgD, IgM, or IgE heavy chain, optionally human. In some embodiments, an antibody heavy chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived. In some embodiments, antibody light chain constant domains contained in the stabilization module are derived from a kappa or lambda light chain, optionally human. In some embodiments, the stabilization module is derived from an antibody light chain constant domain. In some embodiments, an antibody light chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions, and/or deletions) compared to the sequence from which it is derived. In some embodiments, the first and/or second stabilization domains comprise one or more modifications that do not substantially alter their binding affinity for each other. In some embodiments, the first and/or second stabilization domains comprise one or more modifications that increase their binding affinity for each other and/or introduce a non-naturally occurring disulfide bond. In some embodiments, the stabilization module comprises a knob-into-hole modification (see, for example, Carter P. J Immunol Methods. 248:7-15, 2001). For example, in some embodiments, the stabilization module comprises antibody constant domain regions (e.g., CH3 domains) comprising a knob-into-hole modification. In some embodiments, the stabilization module comprises antibody constant domain regions modified by electrostatic steering to enhance their association (see, for example, W02006106905 and Gunasekaran K, et al. J Biol Chem. 285:19637-46, 2010). In some embodiments, the first and second stabilization domains are linked by a disulfide bond.
[0085] Examples of stabilization domains include an Fc region; a hinge region; a CH2 domain; a CH3 domain; a CH4 domain; a CHI domain or CL domain; a TCR constant domain; a leucine zipper domain (e.g., ajun/fos leucine zipper domain, see, e.g., Kostelney et al, J. Immunol, 148: 1547-1553, 1992; or a yeast GCN4 leucine zipper domain); an isoleucine zipper domain; a dimerizing region of a dimerizing cell-surface receptor (e.g., interleukin-8 receptor (IL-8R); or an integrin heterodimer such as LFA-1 or GPIIIb/IIIa); a dimerizing region of a secreted, dimerizing ligand (e.g., nerve growth factor (NGF), neurotrophin-3 (NT- 3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), or brain-derived neurotrophic factor (BDNF); see, e.g., Arakawa et al, J. Biol. Chem. 269:27833-27839, 1994, and Radziejewski et al, Biochem. 32: 1350, 1993); a coiled coil dimerization domain (see, for example, WO2014152878; Fletcher et al, ACS Synth. Biol. 1 :240-250, 2012; and Thomas et al., J. Am. Chem. Soc. 135(13):5161-5166, 2013); and a polypeptide comprising at least one cysteine residue (e.g., from about one, two, or three to about ten cysteine residues) such that disulfide bond(s) can form between the polypeptide and a second polypeptide comprising at least one cysteine residue.
[0086] In some embodiments, the TCRM described herein comprises a first T cell receptor domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule. In some embodiments, both of the TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, both of the TCR-TMs are non-naturally occurring. In some embodiments, the first TCR-TM is derived from one of the transmembrane domains of a T cell receptor (such as an aP TCR or a y5 TCR) and the second TCR-TM is derived from the other transmembrane domain of the T cell receptor. In some embodiments, the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the transmembrane domains of the T cell receptor. Recruitment of TCR-associated signaling molecules can be determined by methods known in the art, such as FACS analysis for TCR-CD3 complex surface expression or co-immunoprecipitation of CD3 subunits with the caTCR. For example, in some embodiments, the first TCR-TM of a TCRM described herein comprises, consists essentially of, or consists of the transmembrane domain of the TCR a chain (e.g., GenBank Accession No: CCI73895) or a variant thereof and the second TCR-TM of the TCRM comprises, consists essentially of, or consists of the transmembrane domain of the TCR P chain (e.g., GenBank Accession No: CCI73893) or a variant thereof. In some embodiments, the first TCR-TM comprises, consists essentially of, or consists of the transmembrane domain of the TCR 5 chain (e.g., GenBank Accession No: AAQ57272) or a variant thereof and the second TCR-TM comprises, consists essentially of, or consists of the transmembrane domain of the TCR y chain (e.g., GenBank Accession No: AGE91788) or a variant thereof. In some embodiments, the first and second TCR-TMs of a TCRM described herein comprise, consist essentially of, or consist of the transmembrane domain of a TCR a chain constant domain (e.g., SEQ ID NO: 35) or a variant thereof and the transmembrane domain of a TCR P chain constant domain (e.g., SEQ ID NO: 36) or a variant thereof, respectively. In some embodiments, the first and second TCR-TMs comprise, consist essentially of, or consist of the transmembrane domain of a TCR 5 chain constant domain (e.g., SEQ ID NO: 37) or a variant thereof and the transmembrane domain of a TCR y chain constant domain (e.g., SEQ ID NO: 38) or a variant thereof, respectively. In some embodiments, the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 39 and 40, or variants thereof, respectively. In some embodiments, the first and second TCR-TMs comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 41 and 42, or variants thereof, respectively. Variants of the transmembrane domains include, without limitation, transmembrane domains with one or more amino acid substitutions compared to the reference sequence. In some embodiments, a variant transmembrane domain comprises no more than 5 amino acid substitutions compared to the reference sequence.
[0087] In some embodiments, the first TCRD further comprises a first connecting peptide amino-terminal to the transmembrane domain and/or the second TCRD further comprises a second connecting peptide amino-terminal to the transmembrane domain. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and/or second connecting peptides comprise, consist essentially of, or consist of all or a portion of the connecting peptide of a TCR a chain constant domain or a variant thereof and all or a portion of the connecting peptide of a TCR P chain constant domain or a variant thereof, respectively. In some embodiments, the first and/or second connecting peptides comprise, consist essentially of, or consist of all or a portion of the connecting peptide of a TCR 5 chain constant domain or a variant thereof and all or a portion of the connecting peptide of a TCR y chain constant domain or a variant thereof, respectively. In some embodiments, the first TCRD further comprises a first TCR intracellular domain carboxy-terminal to the first TCR-TM and/or the second TCRD further comprises a second TCR intracellular domain carboxy-terminal to the second TCR-TM. In some embodiments, the first TCR intracellular domain comprises all or a portion of the intracellular domain of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second TCR intracellular domain comprises all or a portion of the intracellular domain of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first TCRD is a fragment of one chain of a naturally occurring TCR, or a variant thereof, and/or the second TCRD is a fragment of the other chain of the naturally occurring TCR, or a variant thereof. In some embodiments, at least one of the TCRDs is non- naturally occurring. A non-naturally occurring TCR domain may be a corresponding domain of a naturally occurring TCR modified by substitution of one or more amino acids, and/or by replacement of a portion of the corresponding domain with a portion of an analogous domain from another TCR.
[0088] In some embodiments, the first TCR-TM comprises, consists essentially of, or consists of all or a portion of SEQ ID NO: 3, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 3. In some embodiments, the second TCR-TM comprises, consists essentially of, or consists of all or a portion of SEQ ID NO: 4, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 4. In some embodiments, the first and second TCRDs are linked by a disulfide bond. In some embodiments, the first and second TCRDs are linked by a disulfide bond between a residue in the first connecting peptide and a residue in the second connecting peptide. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( . In some embodiments, the TCRM is capable of recruiting each of CD35s, CD3ys, and ( to form a caTCR-CD3 complex (z.e., promotes caTCR-CD3 complex formation).
[0089] In some embodiments, the antigen-binding module is an antibody moiety selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv). In some embodiments, the antigen-binding module is an antibody moiety that is a Fab or a Fab’. In some embodiments, the antigen-binding module is an antibody moiety that is an scFv. In some embodiments, the antibody moiety is fully human, semisynthetic with human antibody framework regions, or humanized.
[0090] In some embodiments, the antigen-binding module comprises a first antigen-binding domain comprising an anti-GPC3 VH antibody domain and a second antigen-binding domain comprising an anti-GPC3 VL antibody domain. In some embodiments, the anti-GPC3 VH antibody domain and anti-GPC3 VL antibody domain CDRs are derived from the same antibody moiety. In some embodiments, some of the anti-GPC3 VH antibody domain and anti-GPC3 VL antibody domain CDRs are derived from different antibody moieties. In some embodiments, the anti-GPC3 VH antibody domain and/or anti-GPC3 VL antibody domain are human, humanized, chimeric, semi -synthetic, or fully synthetic.
[0091] The antigen-binding module in some embodiments is an antibody moiety comprising specific CDR sequences derived from one or more antibody moieties specific to GPC3 (such as a monoclonal antibody) or certain variants of such sequences comprising one or more amino acid substitutions. In some embodiments, the amino acid substitutions in the variant sequences do not substantially reduce the ability of the antigen-binding module to bind to GPC3. Alterations that substantially improve target antigen binding affinity or affect some other property, such as specificity and/or cross-reactivity with related variants of GPC3, are also contemplated. [0092] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module described herein linked to a TCRM described herein, optionally including a stabilization module. For example, in some embodiments, the anti-GPC3 caTCR comprises the antigenbinding module linked to the N-terminus of one or both of the TCRDs. In some embodiments, the anti-GPC3 caTCR comprises a stabilization module between a TCRM and an antigen-binding module. In some embodiments, the anti-GPC3 caTCR further comprises a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length. In some embodiments, the anti-GPC3 caTCR further comprises one or more accessory intracellular domains. In some embodiments, the one or more accessory intracellular domains are carboxy -terminal to the first and/or second TCRD. In some embodiments, the one or more accessory intracellular domains are between the first TCR-TM and the first TCR intracellular domain and/or between the second TCR-TM and the second TCR intracellular domain. In some embodiments, the one or more accessory intracellular domains comprise, individually, a TCR co-stimulatory domain. In some embodiments, the TCR co-stimulatory domain comprises all or a portion of the intracellular domain of an immune co-stimulatory molecule (such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like).
[0093] In some embodiments, the anti-GPC3 caTCR described herein comprises a) an antigen-binding module that specifically binds to GPC3, and b) a TCRM comprising first and second TCR-TMs derived from the transmembrane domains of a TCR (such as an aPTCR or a ySTCR), wherein the TCRM is capable of recruiting at least one TCR-associated signaling molecule. In some embodiments, the antigen-binding module is linked to the amino-terminus of one or more polypeptide chains in the TCRM. For example, in some embodiments, the TCRM comprises two polypeptide chains, and the antigen-binding module is linked to the amino-terminus of one or both of the TCRM polypeptide chains. In some embodiments, the first and second TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first and second TCR-TMs are non-naturally occurring. In some embodiments, the TCRM further comprises at least one connecting peptide or fragment thereof of the TCR amino-terminal to a TCR-TM. In some embodiments, the TCRM further comprises at least one TCR intracellular domain comprising a sequence from an intracellular domain of the TCR carboxy-terminal to a TCR-TM. In some embodiments, the TCRM comprises TCRDs derived from fragments of the TCR chains. In some embodiments, at least one of the TCRDs is non-naturally occurring. In some embodiments, the caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4- IBB (CD137), 0X40, CD30, or CD40) carboxy-terminal to a TCR-TM. In some embodiments, the caTCR lacks a co-stimulatory signaling sequence. In some embodiments, the antigenbinding module is an antibody moiety that specifically binds to GPC3 (“anti-GPC3 antibody moiety”). In some embodiments, the anti-GPC3 antibody moiety comprises an anti-GPC3 VH antibody domain and an anti-GPC3 VL antibody domain. In some embodiments, the anti- GPC3 antibody moiety is human, humanized, chimeric, semi-synthetic, or fully synthetic. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilization module comprises at least one disulfide bond linking the stabilization domains. In some embodiments, the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR- associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( . In some embodiments, the TCRM allows for enhanced recruitment of the at least one TCR- associated signaling molecule as compared to a TCRM comprising the T cell receptor transmembrane domains. In some embodiments, the TCRM promotes caTCR-CD3 complex formation. In some embodiments, there is a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the anti-GPC3 caTCR is a heteromultimer, such as a heterodimer. For example, in some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD, wherein the antigen-binding module is linked to the first and/or second polypeptide chains.
[0094] In some embodiments, the anti-GPC3 caTCR described herein specifically binds GPC3, and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a TCR and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM that is capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen-binding module that specifically binds to GPC3, wherein the antigen-binding module is linked to the first and/or second TCRDs. In some embodiments, both of the TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR- TMs is non-naturally occurring. In some embodiments, the TCR is an aP TCR and the first and second TCR-TMs are derived from TCR a and P subunit transmembrane domains. In some embodiments, the TCR is a y5 TCR and the first and second TCR-TMs are derived from TCR y and 5 subunit transmembrane domains. In some embodiments, the first TCRD further comprises a first TCR connecting peptide or a fragment thereof and/or the second TCRD further comprises a second TCR connecting peptide or a fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR intracellular domain and/or the second TCRD further comprises a second TCR intracellular domain. In some embodiments, the first TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the first TCR-TM is derived and/or the second TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the anti-GPC3 caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4- 1BB (CD137), 0X40, CD30, or CD40). In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first and second stabilization domains are linked by a disulfide bond. In some embodiments, the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( . In some embodiments, the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the T cell receptor transmembrane domains. In some embodiments, the TCRM promotes caTCR-CD3 complex formation. In some embodiments, there is a spacer module between any two anti-GPC3 caTCR modules or domains. . In some embodiments, the antigen-binding module is an antibody moiety (e.g., and anti-GPC3 antibody moiety). In some embodiments, the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
[0095] In some embodiments, the anti-GPC3 caTCR described herein specifically binds GPC3, and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring y5 TCR and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring y5 TCR, wherein the first and second TCRDs form a TCRM that is capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen-binding module that specifically binds to GPC3a, wherein the antigen-binding module is linked to the first and/or second TCRDs. In some embodiments, both of the TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR connecting peptide or a fragment thereof and/or the second TCRD further comprises a second TCR connecting peptide or a fragment thereof. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the first TCR-TM is derived, or a variant thereof, and/or the second connecting peptide comprises all or a portion of the connecting peptide of the TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR intracellular domain and/or the second TCRD further comprises a second TCR intracellular domain. In some embodiments, the first TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the first TCR-TM is derived and/or the second TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the anti-GPC3 caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), 0X40, CD30, or CD40). In some embodiments, the anti- GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first and second stabilization domains are linked by a disulfide bond. In some embodiments, the first and second stabilization domains comprise antibody domains, such as CHI and CL antibody domains, or variants thereof. In some embodiments, the TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD35s, CD3ys, and ( . In some embodiments, the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the naturally occurring y5 T cell receptor transmembrane domains. In some embodiments, the TCRM promotes caTCR-CD3 complex formation. In some embodiments, there is a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the antigen-binding module is an antibody moiety (e.g., and anti-GPC3 antibody moiety). In some embodiments, the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv).
[0096] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antigen-binding module is a Fab.
[0097] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antigen-binding module is a Fab.
[0098] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-GPC3 antibody moiety is a Fab. [0099] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 25. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 24; and b) a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti- GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 24, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antigenbinding module is a Fab.
[0100] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 26, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antigenbinding module is a Fab.
[0101] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 29. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 28, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antigenbinding module is a Fab.
[0102] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR a chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR P chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR P chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR a chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR 5 chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR y chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR y chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR 5 chain. As used herein, the term “fused” means directly fused or indirectly fused.
[0103] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 30; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 31. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31.
[0104] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 5. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5.
[0105] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 63; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 64. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64. [0106] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 65; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 66. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66.
[0107] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 67; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 68. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68.
[0108] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 69; and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence comprising at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 70. In some embodiments, anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, and ii) and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70.
B. Chimeric Co-stimulatory Receptor (CSR) Constructs
[0109] The expression constructs provided herein further encodes a chimeric costimulatory receptor (CSR) that specifically binds to GPC3 (also referred to herein as an “anti-GPC3 CSR”). The anti-GPC3 CSR is capable of stimulating an immune cell on the surface of which it is functionally expressed upon target ligand binding. The anti-GPC3 CSR comprises a ligand-binding module that provides the ligand-binding specificity, a transmembrane module, and a co-stimulatory immune cell signaling module that allows for stimulating the immune cell. The anti-GPC3 CSR lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-GPC3 CSR lacks any primary immune cell signaling sequence. In some embodiments, the anti-GPC3 CSR comprises a single polypeptide chain comprising the ligand-binding module, transmembrane module, and costimulatory signaling module. In some embodiments, the anti-GPC3 CSR comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the ligand-binding module, transmembrane module, and co-stimulatory signaling module. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-GPC3 CSR is a multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond.
[0110] Examples of co-stimulatory immune cell signaling domains for use in the anti- GPC3 CSRs of the invention include the cytoplasmic sequences of co-receptors of the T cell receptor (TCR), which can act in concert with a caTCR (e.g., an anti-GPC3 as described herein) to initiate signal transduction following caTCR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
[OHl] Under some circumstances, signals generated through the TCR alone are insufficient for full activation of the T cell and a co-stimulatory signal is also required. Thus, in some embodiments, T cell activation is mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as “primary T cell signaling sequences”) and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (referred to herein as “co-stimulatory T cell signaling sequences”).
[0112] Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary immune cell signaling sequences include those derived from TCR FcRy, FcRp, CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, and CD66d. A “functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor. “Non-functional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal. The anti-GPC3 CSRs described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an ITAM. In some embodiments, the anti-GPC3 CSRs lack any primary immune cell signaling sequence.
[0113] The co-stimulatory immune cell signaling sequence can be a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4- 1BB (CD137), 0X40, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.
[0114] The target ligand of the anti-GPC3 CSR is GPC3. In some embodiments, the GPC3 is a cell surface-bound GPC3. In some embodiments, the GPC3 target ligand is the same as the GPC3 target antigen of a caTCR (e.g., anti-GPC3 caTCR as described herein) expressed in the same immune cell.
[0115] In some embodiments, the ligand-binding module in the anti-GPC3 CSR is an antibody moiety that specifically binds GPC3 (“anti-GPC3 antibody moiety). In some embodiments, the antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv). In some embodiments, the ligand-binding module is derived from the extracellular domain of a receptor. In some embodiments, the antibody moiety of the anti-GPC3 CSR comprises the CDRs or variables domains (VH and/or VL domains) of an antibody moiety specific for GPC3.
[0116] In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the ligand binding module is a scFv.
[0117] In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the ligand binding module is a scFv. [0118] In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 (such as about any of 1, 2, 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the ligand binding module is a scFv.
[0119] In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 25. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 24; and b) a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 24, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the ligand binding module is a scFv.
[0120] In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 27. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti- GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 26, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the ligand binding module is a scFv.
[0121] In some embodiments, the anti-GPC3 caTCR comprises an antigen-binding module that comprises a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about having at least about 80% (including for example at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 29. In some embodiments, the anti-GPC3 CSR comprises a ligand-binding module that comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti- GPC3 CSR comprises a ligand-binding module that comprises the HC-CDRs of a VH comprising the amino acid sequence of SEQ ID NO: 28, and the LC-CDRs of a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the ligand binding module is a scFv.
[0122] In some embodiments, the transmembrane domain of the anti-GPC3 CSR comprises a transmembrane domain derived from a transmembrane protein including, for example, CD28, CD3s, CD3< CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD30. In some embodiments, the CD30 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 49, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 49. In some embodiments, the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD28. In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 50. In some embodiments, the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD8. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 22.
[0123] In some embodiments, the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from the intracellular domain of a co-stimulatory receptor of a TCR. In some embodiments, the co-stimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of all or a portion of the intracellular domain of a co-stimulatory receptor of, for example, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like. In some embodiments, the co-stimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of all or a portion of the intracellular domain of a co-stimulatory receptor of CD30, CD28, 4-IBB, 0X40, ICOS, CD27, or CD40. In some embodiments, the co-stimulatory immune cell signaling module of the CSR is derived from human CD30. In some embodiments, the co-stimulatory immune cell signaling module derived from human CD30 comprises the amino acid sequence of SEQ ID NO: 44, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 44. [0124] In some embodiments, the co-stimulatory immune cell signaling module of the CSR is derived from human CD28. In some embodiments, the co-stimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 45, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 45. In some embodiments, the co- stimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 46, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 46. In some embodiments, the co-stimulatory immune cell signaling module of the CSR is derived from human 4-IBB. In some embodiments, the co-stimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 47, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 47. In some embodiments, the co-stimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 48, or functional equivalent thereof comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 48.
[0125] In some embodiments, the anti-GPC3 CSR further comprises a spacer module between any of the ligand-binding module, the transmembrane module, and the co- stimulatory signaling module. In some embodiments, the spacer module comprises one or more peptide linkers connecting two anti-GPC3 CSR modules. In some embodiments, the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length.
[0126] In some embodiments, the ligand-binding module (such as an antibody moiety) of the anti-GPC3 CSR specifically binds to GPC3 with a) an affinity that may be at least about 10 (including for example at least about any of 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 750, 1000 or more) times its binding affinity for other molecules; or b) a Kd the may be more than about 1/10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000 or less) times its Kd for binding to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) utilizing, for example, Biacore instruments, or kinetic exclusion assay (KinExA) utilizing, for example, Sapidyne instruments.
[0127] In some embodiments, the anti-GPC3 CSR described herein comprises a) an scFv; and b) a fragment of CD30. In some embodiments, the scFv comprises a VH domain having the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 26, and a VL domain having the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 27. In some embodiments, the scFv comprises a VH domain having the amino acid sequence of SEQ ID NO: 26 and a VL domain having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 33. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the CSR comprises, from amino terminus to carboxy terminus, the scFv, a peptide linker, and the fragment of CD30. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16.
C. c-Jun
[0128] The expression constructs provided herein further encodes a c-Jun polypeptide. [0129] In some embodiments, the c-Jun is a human c-Jun. In some embodiments, the human c-Jun is wildtype human c-Jun. In some embodiments, the human c-Jun has the following amino acid sequence (available at GenBank under accession number AAA59197.1 or at UniProtKB (under accession number P05412.2): MTAKMETTFY DDALNASFLP SESGPYGYSN PKILKQSMTL NLADPVGSLK PHLRAKNSDL LT[S]PDVGLLK LA{S]PELERLI IQSSNGHITT TPTPTQFLCP KNVTDEQEGF AEGFVRALAE LHSQNTLPSV TSAAQPVNGA GMVAPAVASV AGGSGSGGFS ASLHSEPPVY ANLSNFNPGA LSSGGGAPSY GAAGLAFPAQ PQQQQQPPHH LPQQMPVQHP RLQALKEEPQ TVPEMPGETP PLSPIDMESQ ERIKAERKRM RNRIAASKCR KRKLERIARL EEKVKTLKAQ NSELASTANM LREQVAQLKQ KVMNHVNSGC QLMLTQQLQT F ( SEQ ID 1 0 : 1 )
See also Hattori et al., PNAS (1988) 85:9148-52.
[0130] In humans, the c-Jun protein is encoded by the JUN gene, which is located on chromosome 1 (nucleotides 58,780,791 to 58,784,047 of GenBank Accession No. NC_000001.11, minus strand orientation). Synonyms of the JUN gene, and the encoded protein thereof, are known and include "Jun proto-oncogene, AP-1 transcription factor subunit," "v-Jun avian sarcoma virus 17 oncogene homolog," "transcription factor AP-1," "Jun oncogene," "AP-1," "Jun activation domain binding protein," “p39”, and "enhancerbinding protein API." The wild-type human c-Jun protein sequence is 331 amino acids in length.
[0131] In some embodiments, the wildtype human c-Jun comprises at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the wildtype human c-Jun comprises the amino acid sequence of SEQ ID NO: 1.
[0132] In some embodiments, the c-Jun is a mutant c-Jun. In some embodiments, the c-Jun is a mutant human c-Jun. In some embodiments, the mutant c-Jun does not impact the mutant’s ability to rescue dysfunctional (exhausted) T cells. In some embodiments, the mutant c-Jun comprises at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, or 99%) sequence identity with the C-terminal amino acid residues (e.g., C-terminal 50, 75, 100, 150, 200, 250, or more, amino acid residues), the C-terminal portion (e.g., quarter, third, or half) or C-terminal domains (e.g., epsilon, bZIP, and amino acids C-terminal thereof) of a wildtype c-Jun. In some embodiments, the N-terminal amino acid residues (e.g., N-terminal 50, 75, 100, or 150 or more), the N-terminal portion (e.g., quarter, third, or half) or N- terminal domains (e.g., delta, transactivation domain, and amino acids N-terminal thereof) of a wildtype c-Jun are deleted, mutated, or otherwise inactivated.
[0133] In some embodiments, the c-Jun comprises one or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating substitutions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating deletions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating insertions in its transactivation domain. In some embodiments, the c-Jun comprises one or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its delta domain. In some embodiments, the c-Jun comprises one or more inactivating substitutions in its delta domain. In some embodiments, the c-Jun comprises one or more inactivating deletions in its delta domain. In some embodiments, the c-Jun comprises one or more inactivating insertions in its delta domain. In some embodiments, the c-Jun comprises or more inactivating mutation(s) (e.g., substitutions, deletions, or insertions) in its transactivation domain and its delta domain.
[0134] In some embodiments, the c-Jun comprises a serine (S) to alanine (A) substitution. In some embodiments, the c-Jun comprises one or both of S63 A and S73 A substitutions as compared to SEQ ID NO: 1 (the positions are boxed in the amino acid sequence of human c- Jun above). In some embodiments, the c-Jun has a deletion between amino acid residues 2 and amino acid residues 102 or between residues 30 and 50 as compared to SEQ ID NO: 1. [0135] In certain embodiments, the anti-GPC3 caTCR and/or the anti-GPC3 CSR and/or the c-Jun polypeptides further comprise an affinity or purification tag available for various use purposes; for example, it may be used to enhance the purification efficiency of the target polypeptide. In one embodiment, the tag is a myc, HIS or HA tag.
[0136] In some aspects, an immune cell described herein has been modified to comprise an exogenous nucleotide sequence encoding a c-Jun polypeptide, wherein the exogenous nucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 52-62. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 52-62. [0137] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 52. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 52. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 52.
[0138] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 53. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 53. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 53.
[0139] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 54. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 54. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 54.
[0140] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 55. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% to the nucleic acid sequence set forth in SEQ ID NO: 55. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 55.
[0141] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 56. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 56. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 56. [0142] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 57. In some aspects, the exogenous polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 57.
[0143] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 58. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 58. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 58.
[0144] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 59. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 59. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 59. [0145] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 60. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 60. In some aspects, the exogenous polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 60.
[0146] In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 61. In some aspects, an exogenous polynucleotide encoding a c-Jun polypeptide has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 61. In some aspects, the exogenous nucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 61.
[0147] Exemplary c-Jun nucleotide sequences are provided in the Sequence Listing provided herein.
[0148] The c-Jun nucleotide sequence disclosed herein can be codon-optimized using any methods known in the art. For instance, in some aspects, the codons of a c-Jun nucleotide sequence disclosed herein has been optimized to modify (e.g., increase or decrease) one or more of the following parameters compared to the wild-type nucleotide sequence (e.g., SEQ ID NO: 62): (i) codon adaptation index (z.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and unstable motifs; (iv) repeat sequences (e.g., direct repeats, inverted repeats, dyad repeats); (v) restriction enzyme recognition sites; or (vi) combinations thereof.
[0149] While certain disclosures provided herein generally relate to modifying an immune cell to comprise an exogenous nucleotide sequence encoding a c-Jun protein (wild-type c-Jun or a variant thereof), it will be apparent to those skilled in the art that other suitable methods can be used to induce and/or increase c-Jun protein expression (either wild-type or a variant thereof) in a cell. For instance, as described herein, in some aspects, the endogenous c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa). Unless indicated otherwise, disclosures provided above using exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa).
[0150] In some aspects, due to the modification (e.g., introduction of the exogenously introduced c-Iun nucleotide sequence and/or transcriptional activator), the engineered cells overexpress, i.e., express a higher level (e.g., at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% more, or at least about 1.5-, 2-, 3-, 4-, 5-, or 10-fold more) of, a c-Iun protein than corresponding cells without such a modification ("reference cell"). The terms "express increased levels [or amounts] of," "overexpress," or have "increased expression of' (and similar forms of the phrase used herein), are used interchangeably.
[0151] In some aspects, the engineered (or modified) cells described herein express at least about 2-100 fold more, about 5-50 fold more, about 5-40 fold more, about 5-30 fold more, about 5-20 fold more, about 8-20 fold more, or about 10-20 fold more c-Iun protein than the reference cell. In some aspects, the expression of the c-Iun protein in a modified cell described herein is increased by at least about 0.5-fold, by at least about 1-fold, by at least about 2-fold, by at least about 3-fold, by at least about 4-fold, by at least about 5-fold, by at least about 6-fold, by at least about 7-fold, by at least about 8-fold, by at least about 9-fold, by at least about 10-fold, by at least about 12-fold, by at least about 14-fold, by at least about 16-fold, by at least about 18-fold, by at least about 20-fold, by at least about 25-fold, by at least about 30-fold, by at least about 35-fold, by at least about 40-fold, by at least about 45- fold, or by at least about 50-fold, compared to the expression of the c-Iun protein in the reference cell.
[0152] In some aspects, the increased expression of the c-Iun protein can improve and/or enhance one or more properties of the modified immune cells (e.g., T cells, such as CD4+ and/or CD8+ T cells). Non-limiting examples of such properties include: resistance to exhaustion (e.g., as indicated by reduced expression of exhaustion markers, such as PD-1, CD39, TIM-3, and/or LAG-3; increased persistence/survival; delay of the onset of dysfunctional states; and/or increased cytokine (e.g., IFN-y and/or IL-2) production), increased expansion/proliferation, increased antigen sensitivity, improved effector function, in particular, improved effector function following repeated antigen stimulation (e.g., cytokine production upon antigen stimulation, lysis of cells expressing the target antigen, or both), or combinations thereof.
[0153] Assays useful for measuring exhaustion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, cytokine production/release, and gene expression profiles are known in the art and include, for example flow cytometry, intracellular cytokine staining (ICS), INCUCYTE® immune cell killing analysis, Meso Scale Discovery (MSD, a platform which combines electrochemiluminescence and MULTI-ARRAY technology) or similar assay, persistent antigen stimulation assays, bulk and single cell RNAseq (see e.g., Front Genet. 2020; 11 :220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci. 2: 139-173), cytotoxicity /killing assays, ELISA, western blot and other standard molecular and cell biology methods such as described herein or as described, for example, in Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021) or elsewhere.
D. Nucleic Acids
[0154] The anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be introduced to the T cells or progenitor cells through one or more nucleic acid molecules (e.g., DNA or RNA such as mRNA). In some embodiments, the nucleic acid molecules may be placed on one or more DNA or RNA vectors for introduction into the host cells.
[0155] The nucleic acid molecules (e.g., DNA or RNA vectors containing them) may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). Alternatively, the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules. Examples of viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus.
[0156] In some embodiments, the coding sequences for the two polypeptide chains of the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be placed on a single expression construct. The four coding sequences may be placed into one or more expression cassettes on the construct, each cassette being its own transcription unit (e.g., with its own promoter and polyadenylation site and other transcription control elements). In particular embodiments, the four coding sequences may be placed into a single expression cassette (e.g., a polycistronic expression cassette), with the four coding sequences being transcribed under a common promoter. In a polycistronic arrangement, the coding sequences are in-frame and separated from each other by the coding sequence of a self-cleaving peptide (e.g., a 2A self-cleaving peptide such as a T2A, P2A, E2A, or F2A peptide). Alternatively, the coding sequences may be separated from each other by a ribosomal internal entry site (IRES). Thus, the polycistronic (e.g., quad-cistronic) expression cassette is transcribed into a single RNA but ultimately the single RNA is processed and translated into separate polypeptides.
[0157] In some embodiments, the polycistronic expression cassette comprises coding sequences for the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun. In some embodiments, the expression cassette comprises coding sequences for the antigen binding module of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 28 and 29. In some embodiments, the expression cassette comprises coding sequences for a TCRM derived from a human y/8 TCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 4 and 5. In some embodiments, the expression cassette comprises coding sequences for the two polypeptide chains of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 30 and 31. In some embodiments, the expression cassette comprises coding sequences for the amino acid sequences of SEQ ID NOs: 10 and 5. In some embodiments, the expression cassette comprises a coding sequence for the ligand binding module of the anti-GPC3 CSR. In some embodiments, the expression cassette comprises a coding sequence for the amino acid sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for a partial sequence of human CD30 of the anti-GPC3 CSR. In some embodiments, the expression cassette comprises a coding sequence for the amino acid sequence of SEQ ID NO: 43. In some embodiments, the expression cassette comprises a coding sequence for the human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and a coding sequence for SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 30 and 31, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 10 and 5, and coding sequences for SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette further comprises a coding sequence for a signal peptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 2.
[0158] The expression cassettes (polycistronic or monocistronic) may contain a promoter that is constitutively active in mammalian (e.g., human or human T) cells. Such promoters include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor- la (EF-la) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Core or minimal promoters derived from the aforementioned promoters also are contemplated. Alternatively, the expression cassettes may comprise an inducible promoter system. Exemplary inducible promoter systems include, without limitation, hormone-regulated elements, synthetic ligand- regulated elements, ionizing radiation-regulated elements, tetracycline (Tet) systems (e.g., “Tet-Off” and “Tet-On” systems), and NF AT systems (see, e.g., Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics. (2018) 12: 16-25). In some embodiments, the expression cassettes include a signal peptide. In some embodiments, the signal peptide comprises the coding sequence for the amino acid sequence of SEQ ID NO: 2.
[0159] In some embodiments, the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and/or translation of the coding sequences. For example, a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3 ’ untranslated region of the expression cassette.
[0160] In the expression cassettes, the transcription/translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts.
[0161] In certain embodiments, the present disclosure provides a single-vector construct (e.g., a lentiviral vector) comprising a polycistronic expression cassette, comprising a mammalian promoter, a c-Jun coding sequence, coding sequences for the two anti-GPC3 caTCR chains, a coding sequence for the anti-GPC3 CSR, and a polyadenylation signal sequence. The coding sequences are linked by a nucleotide linker that may be an IRES or a coding sequence for a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A, or functional equivalents thereof). By way of example, FIG. 1A illustrates such an expression cassette, wherein the promoter is an EF- la promoter.
[0162] In particular embodiments, the expression cassette encodes a c-Jun comprising SEQ ID NO: 1, an anti-GPC3 caTCR comprising two polypeptide chains comprising SEQ ID NOs: 30 and 31, respectively, and an anti-GPC3 CSR comprising SEQ ID NO: 33 (e.g., Construct 2 in FIG. 1A). The construct may be a recombinant lentiviral vector and may further comprise a central polypurine tract (cPPT) upstream of the EF-la promoter, and a WPRE sequence between the CSR coding sequence and an SV40 polyadenylation signal, or other sequences for efficient transduction and expression in mammalian cells.
[0163] The coding sequences in the expression cassettes may be codon-optimized for optimal expression levels in a host cell of interest (e.g., human cells).
[0164] The nucleic acid molecules encoding the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun may be integrated into the genome of the engineered cells or remain episomal. The integration may be targeted integration occurring through gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases, and meganucleases).
[0165] The engineered cells can be enriched for by positive selection techniques. For example, the cells can be selected for their ability to bind to GPC3 in, e.g, flow cytometry assays. To confirm c-Jun expression, RT-PCT may be performed on the engineered T cells. The positive selection may lead to enrichment of caTCR+CSR+c-Jun+ cells in a cell population, where the triple positive T cells constitute more than 30, 35, 40, 45 ,50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% of the total cell population. The engineered cells may be cryopreserved until use.
E. Variants
[0166] In some embodiments, amino acid sequence variants of the anti-GPC3 caTCR, the anti-GPC3 CSR, and the c-Jun provided herein are contemplated. In some embodiments, amino acid sequence variants of the antigen binding module of the anti-GPC3 caTCR and/or the ligand binding module of the anti-GPC3 CSR are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the one or more of the polypeptides encoded by the expression constructs (e.g., the anti-GPC3 caTCR, anti- GPC3 CSR, and/or c-Jun). Amino acid sequence variants of an anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., GPC3-binding.
[0167] In some embodiments, anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs (hypervariable regions) and FRs (framework regions).
[0168] Conservative substitutions are shown in Table 2 below.
Table 2. Conservative Substitutions.
Figure imgf000065_0001
Figure imgf000066_0001
[0169] Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; and f. aromatic: Trp, Tyr, Phe.
[0170] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
[0171] An exemplary substitutional variant is an affinity-matured antibody moiety comprised of the antigen binding module of the anti-GPC3 caTCR or the ligand binding module of the anti-GPC CSR, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties are displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody moiety affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury , Methods Mol. Biol. 207: 179-196 (2008)), and/or specificity determining residues (SDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).
[0172] In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody moiety variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
[0173] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the anti- GPC3 caTCR and/or the anti-GPC3 CSR to bind GPC3. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In some embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
[0174] A useful method for identification of residues or regions of an antibody (e.g., an antibody moiety comprised in the antigen binding module of the anti-GPC3 caTCR or the anti-GPC3 CSR) that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be determined to identify contact points between the anti-GPC3 caTCR and/or the anti-GPC3 CSR and GPC3. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
Variants may be screened to determine whether they exhibit the desired properties.
[0175] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody moiety (e.g, an antigen binding module of the anti- GPC3 caTCR and/or a ligand binding module of the anti-GPC3 CSR) with an N-terminal methionyl residue. Other insertional variants of the antibody moiety include the fusion to the N- or C-terminus of the antibody moiety to an enzyme (e.g for ADEPT, antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the construct (e.g., the anti-GPC3 caTCR and/or anti-GPC3 CSR construct).
Glycosylation Variants
[0176] In some embodiments, an anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun provided herein is altered to increase or decrease the extent to which the expression construct is glycosylated. Addition or deletion of glycosylation sites to an expression construct may be conveniently accomplished by altering the amino acid sequence of the anti-GPC3 caTCR, the anti-GPC3 CSR, and/or c-Jun construct or polypeptide portion thereof such that one or more glycosylation sites is created or removed.
[0177] Where the anti-GPC3 caTCR comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-GPC3 construct of the invention may be made in order to create anti-GPC3 construct variants with certain improved properties.
[0178] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum. N-glycosylation of the Fc is important for binding to FcyR; and afucosylation of the N-glycan increases Fc's binding capacity for FcyRIIIa. Increased FcyRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications in which cytotoxicity is desirable. [0179] In some embodiments, an enhanced effector function can be detrimental when remediated cytotoxicity is undesirable. In some embodiments, the Fc fragment or CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.
Cysteine Engineered Variants
[0180] In some embodiments, it may be desirable to create cysteine engineered anti-GPC3 caTCR constructs, anti-GPC3 CSR constructs, and/or c-Jun polypeptides, in which one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the constructs described herein. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the construct and may be used to conjugate the anti-GPC3 construct to other moieties. Cysteine engineered expression constructs may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
III. Immune Cells
[0181] The present invention in one aspect provides modified cells (such as immune cells, for example T cells) expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a human c-Jun polypeptide, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and a human c-Jun polypeptide described herein. Exemplary methods of preparing modified cells (such as T cells) expressing the anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptides, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and a human c-Jun polypeptide described herein, are provided herein.
[0182] In some embodiments, the present invention provides an immune cell (such as a T cell) presenting on its surface a caTCR, an CSR, and a c-Jun polypeptide according to any of the caTCRs, CSRs, and c-Jun polypeptides described herein described herein (such an immune cell is also referred to herein as an “caTCR plus CSR plus c-Jun immune cell”). The caTCRs and CSRs of the present invention specifically target GPC3 (such caTCRs and CSRs are also referred to herein as “anti-GPC3 caTCRs” and “anti-GPC3 CSRs”, respectively). In some embodiments, the immune cell comprises nucleic acid encoding the anti-GPC3 caTCR and anti-GPC3 CSR, wherein the anti-GPC3 caTCR and anti-GPC3 CSR are expressed from the nucleic acid and localized to the immune cell surface. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the T cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of the endogenous TCR subunits of the immune cell. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or P chains or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. Modifications of cells to disrupt gene expression include any such techniques known in the art, including for example RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR- or TALEN-based gene knockout), and the like.
[0183] For example, in some embodiments, there is provided an immune cell (such as a T cell) comprising nucleic acid encoding an anti-GPC3 caTCR according to any of the anti- GPC3 caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptide are expressed from the nucleic acid and localized to the immune cell surface. In some embodiments, the nucleic acid comprises a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of the anti-GPC3 caTCR, an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR, and a human c-Jun nucleic acid sequence encoding a human c- Jun polypeptide chain. In some embodiments, the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and human c-Jun nucleic acid sequences are each contained in different vectors. In some embodiments, some or all of the nucleic acid sequences are contained in the same vector. Vectors may be selected, for example, from the group consisting of mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses). In some embodiments, one or more of the vectors is integrated into the host genome of the immune cell. In some embodiments, the first and second anti-GPC3 caTCR nucleic acid sequences, the anti-GPC3 CSR nucleic acid sequence, and the human c-Jun nucleic acid sequence are each under the control of different promoters. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
[0184] Thus, in some embodiments, there is provided an anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun (“caTCR+CSR+c-Jun”) immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the anti-GPC3 caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; c) an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; and d) a c-Jun nucleic acid sequence encoding a c-Jun polypeptide, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form the anti-GPC3 caTCR, wherein the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid to form the anti-GPC3 CSR, wherein the c- Jun polypeptide is expressed from the c-Jun nucleic acid to form to c-Jun polypeptide, and wherein the anti-GPC3 caTCR and anti-GPC3 CSR localize to the surface of the immune cell. In some embodiments, the first anti-GPC3 caTCR nucleic acid sequence is contained in a first vector (such as a lentiviral vector), the second anti-GPC3 caTCR nucleic acid sequence is contained in a second vector (such as a lentiviral vector), the anti-GPC3 CSR nucleic acid sequence is contained in a third vector (such as a lentiviral vector), and the c-Jun nucleic acid sequences is contained in a fourth vector (such as a lentiviral vector). In some embodiments, some or all of the first and second anti-GPC3 caTCR nucleic acid sequences and anti-GPC3 CSR nucleic acid sequence are contained in the same vector (such as a lentiviral vector). In some embodiments, some or all of the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and c-Jun nucleic acid sequence are contained in the same vector (such as a lentiviral vector). In some embodiments, each of the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequence, and c-Jun nucleic acid sequence are, individually, operably linked to a promoter. In some embodiments, some or all of the nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors). In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.
[0185] In some embodiments, there is provided a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second vector comprising a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; c) a third vector comprising a third promoter operably linked to an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; and d) a fourth vector comprising a fourth promoter operatively linked to a c-Jun nucleic acid sequence encoding the c-Jun polypeptide, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form the anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form the anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence to form the c-Jun polypepide, and wherein the caTCR and CSR localize to the surface of the immune cell. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell.
[0186] In some embodiments, there is provided a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR and ii) a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second vector comprising a third promoter operably linked to an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; and c) a third vector comprising a fourth promoter operably linked to a c-Jun nucleic acid sequence encoding a c-Jun polypeptide, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form the anti-GPC3 caTCR, the anti- GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form the anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR localizes to the surface of the immune cell. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cell. It is to be appreciated that embodiments where any of the nucleic acid sequences are swapped are also contemplated, such as where the first or second anti- GPC3 caTCR nucleic acid sequence is swapped with the anti-GPC3 CSR nucleic acid sequence or the c-Jun nucleic acid sequence.
[0187] In some embodiments, there is provided a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR and ii) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, wherein the first and second anti-GPC3 caTCR nucleic acid sequences are under the control of a first promoter; b) a second vector comprising a second promoter operably linked to an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; and c) a third vector comprising a third promoter operably linked to a c-Jun nucleic acid sequence encoding a c-Jun polypeptide, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form the anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form the anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR and anti- GPC3 CSR localize to the surface of the immune cell. In some embodiments, the first promoter is operably linked to the 5’ end of the first anti-GPC3 caTCR nucleic acid sequence, and there is nucleic acid linker selected from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3’ end of first anti-GPC3 caTCR nucleic acid sequence to the 5’ end of the second anti-GPC3 caTCR nucleic acid sequence, wherein the first anti-GPC3 caTCR nucleic acid sequence and the second anti-GPC3 caTCR nucleic acid sequence are transcribed as a single RNA under the control of the promoter. In some embodiments, the first promoter is operably linked to the 5’ end of the second anti-GPC3 caTCR nucleic acid sequence, and there is nucleic acid linker selected from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A) linking the 3’ end of second anti-GPC3 caTCR nucleic acid sequence to the 5’ end of the first anti-GPC3 caTCR nucleic acid sequence, wherein the first anti-GPC3 caTCR nucleic acid sequence and the second anti-GPC3 caTCR nucleic acid sequence are transcribed as a single RNA under the control of the promoter. In some embodiments, the first and/or second promoters have the same sequence. In some embodiments, the first and/or second promoters have different sequences. In some embodiments, the first and/or second promoters are inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR- TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of the immune cell. It is to be appreciated that embodiments where any of the nucleic acid sequences are swapped are also contemplated, such as where the first or second anti-GPC3 caTCR nucleic acid sequence is swapped with the anti-GPC3 CSR nucleic acid sequence or the c-Jun nucleic acid sequence.
[0188] In some embodiments, there is provided a caTCR+CSR+c-Jun immune cell (such as a T cell) expressing on its surface an anti-GPC3 caTCR according to any of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c- Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a vector comprising a) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; c) an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; and d) a c- Jun nucleic acid sequence encoding a c-Jun polypeide, wherein the first and second anti- GPC3 caTCR nucleic acid sequences, the anti-GPC3 CSR nucleic acid sequence, and the c- Jun polypeptide are under the control of a single promoter; wherein the first and second anti- GPC3 caTCR polypeptide chains are expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form the anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form the anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti- GPC3 caTCR and anti-GPC3 CSR localize to the surface of the immune cell. In some embodiments, the promoter is operably linked to one of the nucleic acid sequences, which is linked to the other nucleic acid sequences by nucleic acid linkers selected, individually, from the group consisting of an internal ribosomal entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A), such that the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are transcribed as a single RNA under the control of the promoter. In some embodiments, the promoter is inducible. In some embodiments, the immune cell does not express the TCR subunits from which the TCR-TMs of the anti-GPC3 caTCR are derived. For example, in some embodiments, the immune cell is an aP T cell and the TCR-TMs of the introduced anti-GPC3 caTCR comprise sequences derived from TCR 5 and y chains, or the immune cell is a y5 T cell and the TCR- TMs of the introduced caTCR comprise sequences derived from TCR a and P chains. In some embodiments, the immune cell is modified to block or decrease the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an aP T cell modified to block or decrease the expression of the TCR a and/or p chains, or the immune cell is a y5 T cell modified to block or decrease the expression of the TCR y and/or 5 chains. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell. In some embodiments, the vector is a viral vector (such as a lentiviral vector) integrated into the host genome of the immune cell.
A. Immune Cell Sources
[0189] The source of the engineered immune cells of the present disclosure may be a patient to be treated (z.e., autologous cells) or from a donor who is not the patient to be treated (e.g., allogeneic cells). In some embodiments, the engineered immune cells are engineered T cells. The engineered T cells herein may be CD4+CD8‘ (z.e., CD4 single positive) T cells, CD4'CD8+ (z.e., CD8 single positive) T cells, or CD4+CD8+ (double positive) T cells. Functionally, the T cells may be cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells, or a mixture thereof. The T cells to be engineered may be autologous or allogeneic.
[0190] Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the present invention, any number of T cell lines available in the art may be used. In some embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
[0191] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3/anti-CD28 (z.e., 3*28)- conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In some embodiments, the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In some embodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield.
Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such as in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
[0192] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD- 14, CD20, CD1 lb, CD-16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar methods of selection.
[0193] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in some embodiments, a concentration of about 2 billion cells/ml is used. In some embodiments, a concentration of about 1 billion cells/ml is used. In some embodiments, greater than about 100 million cells/ml is used. In some embodiments, a concentration of cells of about any of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In some embodiments, a concentration of cells of about any of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In some embodiments, a concentration of about 125 or about 150 million cells/ml is used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
[0194] In some embodiments of the present invention, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in some embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
[0195] Whether prior to or after genetic modification of the T cells to express a desirable expression construct, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.
[0196] Generally, the T cells of the invention 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-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated, such as by contact with an anti-CD3 antibody, or antigenbinding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc . 30(8):3975-3977, 1998; Haanen et aL, J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(1- 2):53-63, 1999).
[0197] The cell culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the culture conditions include addition of IL-2, IL-7 and/or IL-15.
[0198] In some embodiments, the cells to be engineered may be pluripotent or multipotent cells that are differentiated into mature T cells after engineering. These non-T cells may be allogeneic and may be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem or progenitor cells. For ease of description, pluripotent and multipotent cells are collectively called “progenitor cells” herein.
[0199] Where allogeneic cells are used, they are preferably engineered to reduce graft- versus-host rejection (e.g., by knocking out the endogenous B2M and/or TRAC genes).
B. Engineering of Immune or Progenitor Cells
[0200] In some aspects, cellular engineering of an immune cell comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., an anti-GPC3 caTCR and anti-GPC3 CSR), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell, function (e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression compared to a corresponding cell that has not been modified), or any combination thereof. As further described elsewhere in the present disclosure, in some aspects, a cell can be engineered or modified with a transcription activator (e.g., CRISPR/Cas system-based transcription activator), wherein the transcription activator is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun).
[0201] In some aspects, a cell described herein has been modified with a transcriptional activator, which is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As used herein, the term “transcriptional activator” refers to a protein that increases the transcription of a gene or set of genes (e.g., by binding to enhancers or promoter-proximal elements of a nucleic acid sequence and thereby inducing its transcription). Non-limiting examples of such transcriptional activators that can be used with the present disclosure include: Transcription Activator-like Effector (TALE)-based transcriptional activator, zinc finger protein (ZFP)-based transcriptional activator, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system-based transcriptional activator, or a combination thereof. See, e.g., Kabadi et al., Methods (2014) 69(2): 188-197, which is incorporated herein by reference in its entirety.
[0202] In some aspects, a cell described herein has been modified with a CRISPR/Cas- system-based transcriptional activator, such as CRISPR activation (CRISPRa). See, e.g., Nissim et al., Molecular Cell (2014) 54(4):698-710; Perez-Pinera et al., Nat. Methods (2013) 10(10):973-976; Maeder et al., Nat. Methods (2013) 10(10):977-979; Cheng et al., Cell Res. (2013) 23(10): 1163-71; Farzadfard et al., ACS Synth. Biol. (2013) 2(10):604-13; all of which are incorporated herein by reference in their entirety. CRISPRa is a type of CRISPR tool that comprises the use of modified Cas proteins that lack endonuclease activity but retain the ability to bind to its guide RNA and the target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins which can be used with the present disclosure are known in the art (see, e.g., Pandelakis et al., Cell Systems (2020) 10(1): 1 - 14, which is incorporated herein by reference in its entirety). In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to in the art as “dCas9”). In some aspects, the modified Cas protein comprises a modified Casl2a protein. In some aspects, a modified Cas protein that is useful for the present disclosure is bound to a guide polynucleotide (e.g., small guide RNA) (“modified Cas-guide complex”), wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In certain aspects, the guide polynucleotide comprises a recognition sequence that is complementary to the promoter region of an endogenous nucleic acid sequence encoding a protein of interest. In some aspects, one or more transcriptional activators are attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when the modified Cas-guide complex is introduced into a cell, the one or more transcription activators can bind to a regulatory element (e.g., a promoter region) of an endogenous gene and thereby induce and/or increase the expression of the encoded protein (e.g., c-Jun). Illustrative examples of common general activators that can be used include the omega subunit of RNAP, VP16, VP64 and p65 (see, e.g., Kabadi and Gersbach, Methods (2014) 69(2): 188-97). [0203] In some aspects, one or more transcriptional repressors (e.g., Kruppel-associated box domain (KRAB)) can be attached to the modified Cas-guide complex (e.g., the N- and/or C-terminus of the modified Cas protein), such that when introduced into a cell, the one or more transcriptional repressors can repress or reduce the transcription of a gene, e.g., such as those that can interfere with the expression of c-Jun (e.g., Bach2). See, e.g., US20200030379A1 and Yang et al., J Transl Med. (2021) 19:459, each of which is incorporated herein by reference in its entirety. In some aspects, a modified Cas protein useful for the present disclosure can be attached to both one or more transcriptional activators and one or more transcriptional repressors.
[0204] Not to be bound by any one theory, in some aspects, the use of such modified Cas proteins can allow for the conditional transcription and expression of a gene of interest. For example, in some aspects, a cell (e.g., T cells) is modified to comprise a recombinant antigen receptor (e.g., an anti-GPC3 caTCR and an anti-GPC3 CSR), which is linked to a protease (e.g., tobacco etch virus (TEV)) and a single guide RNA (sgRNA) targeting the promoter region of c-Jun. In some aspects, the cell is modified to further comprise a linker for activation of T cells (LAT), complexed to the modified Cas protein attached to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., TEV-cleavable linker). Upon activation of the antigen receptor, the modified Cas protein is released for nuclear localization and conditionally and reversibly induces the expression of c-Jun. See, e.g., Yang et al., J Immunother Cancer (2021) 9(Suppl2):A164, which is herein incorporated by reference in its entirety.
[0205] As will be apparent to those skilled in the art, in some aspects, a cell described herein has been modified using a combination of approaches. For instance, in some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding one or more proteins (e.g, an anti-GPC3 caTCR, an anti-GPC3 CSR, and a truncated EGFR (EGFRt)) and (ii) an exogenous transcriptional activator (e.g, CRISPRa) that increases expression of an endogenous protein (e.g., c-Jun). In some aspects, a cell has been modified to comprise (i) an exogenous nucleotide sequence encoding a first protein (e.g., an anti-GPC3 caTCR), (ii) an exogenous nucleotide sequence encoding a second protein (e.g., an anti- GPC3 CSR), and (iii) an exogenous nucleotide sequence encoding a protein (e.g., a c-Jun protein). In some aspects, the modified cell can further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first, second, and third proteins can be part of a single polycistronic vector.
[0206] Unless indicated otherwise, the one or more exogenous nucleotide sequences and/or transcriptional activators can be introduced into a cell using any suitable methods known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to a cell include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.
[0207] In some aspects, a cell has been modified with a transcriptional activator (e.g., CRISPR/Cas-system-based transcription activator, e.g., CRISPRa), such that the expression of the endogenous c-Jun protein is increased compared to a corresponding cell that has not been modified with the transcriptional activator.
[0208] While certain disclosures provided herein generally relate to modifying an immune cell to comprise an exogenous nucleotide sequence encoding a c-Jun protein (wild-type c-Jun or a variant thereof), it will be apparent to those skilled in the art that other suitable methods can be used to induce and/or increase c-Jun protein expression (either wild-type or a variant thereof) in a cell. For instance, as described herein, in some aspects, the endogenous c-Jun protein expression can be increased with a transcriptional activator (e.g., CRISPRa). Unless indicated otherwise, disclosures provided herein using exogenous nucleotide sequences equally apply to other approaches of inducing and/or increasing c-Jun protein expression in a cell provided herein (e.g., transcriptional activator, e.g., CRISPRa).
[0209] The immune cells (e.g., T cells) or progenitor cells herein may be engineered to express an anti-GPC3 caTCR and an anti-GPC3 CSR, and overexpress c-Jun (e.g., a human c-Jun polypeptide). The anti-GPC3 caTCR may specifically bind to GPC3 tumor cell, and the anti-GPC3 CSR may bind specifically GPC3 (e.g., the same GPC3 but a different GPC3 epitope) on the tumor cell. As used herein, a receptor (e.g., an anti-GPC3 caTCR or an anti- GPC3 CSR) is said to specifically bind to GPC3 when the KD for the binding is < 100 nM, e.g., < 10 nM or < 1 nM. A KD binding affinity constant can be measured, e.g., by surface plasmon resonance (using, e.g., a Biacore™ or Octet™ system).
[0210] In some embodiments, the immune cell expresses the anti-GPC3 caTCR with even cell surface distribution. In some embodiments, the immune cell expresses the anti-GPC3 CSR with even cell surface distribution. Even cell surface distribution can be characterized, for example, by staining patterns with continuous appearance and even thickness or signal intensity. For example, in some embodiments, a composition, such as a pharmaceutical composition, comprising anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun immune cells comprises fewer than about 10 % (such as fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) cells with aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface. Aggregation can be characterized, for example, by staining patterns with uneven thickness or signal intensity, or discontinuous, lumpy, punctate, and/or uneven distribution patterns. In some embodiments, the caTCR+CSR+c-Jun T cell expresses the anti-GPC3 caTCR and/or anti-GPC3 CSR with less than about 10 % (such as less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface. In some embodiments, the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell expresses the anti-GPC3 caTCR and/or anti-GPC3 CSR with less than about 10 % (such as less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 %) aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface. In some embodiments, the anti-GPC3 caTCR plus anti- GPC3 CSR plus human c-Jun T cell has a low level of antigen-independent anti-GPC3 caTCR and/or anti-GPC3 CSR activation. In some embodiments, the anti-GPC3 caTCR T cell has a low level of antigen-independent anti-GPC3 caTCR and/or anti-GPC3 CSR activation. In some embodiments, the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c- Jun T cell has a low level of T cell exhaustion. T cell exhaustion naturally occurs during conditions of extended immune activation, such as with cancer or chronic infection, where T cells become dysfunctional. T cell exhaustion may be characterized by impaired effector function, prolonged expression of inhibitory receptors, and/or an altered transcriptional state compared to functional effector or memory T cells. Optimal clearance of tumor cells and infections is prevented by T cell exhaustion. T cell exhaustion of the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell can be characterized by any means known in the art, for example, by determining its functional and/or phenotypic profile (Wherry, E. J., Nature immunology 12(6): 492-499, 2011; Jiang, Y., et al., Cell death & disease 6(6): el792, 2015). For example, in some embodiments, the anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell expresses low levels of one or more markers of T cell exhaustion, including, for example, PD-1, LAG-3, TIM-3, CTLA-4, and BTLA. In some embodiments, anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cell maintains levels characteristic of nonexhausted T cells for IL-2 production, TNF-a production, IFN-y production, and granzyme B production, and/or maintains ex vivo killing capacity in the presence of target cells, suggesting that caTCR+CSR+c-Jun T cell is not undergoing self-activation and premature exhaustion.
IV. Pharmaceutical Compositions
[0211] Also provided herein are anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun (“caTCR+CSR+c-Jun”) compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising an immune cell (such as a T cell) presenting on its surface an anti-GPC3 caTCR according to any of the anti-GPC3 caTCRs described herein, an anti- GPC3 CSR according to any of the CSRs described herein, and a c-Jun polypeptide. In some embodiments, the caTCR+CSR+c-Jun immune cell composition is a pharmaceutical composition.
[0212] The composition may comprise a homogenous cell population comprising caTCR+CSR+c-Jun immune cells of the same cell type and expressing the same anti-GPC3 caTCR, anti-GPC3 CSR, and c-Jun, or a heterogeneous cell population comprising a plurality of caTCR+CSR+c-Jun immune cell populations comprising caTCR+CSR+c-Jun immune cells of different cell types, expressing different anti-GPC3 caTCRs, expressing different anti-GPC3 CSRs, and/or expressing different c-Jun polypeptides (e.g., wildtype or mutant c- Jun polypeptides). The composition may further comprise cells that are not caTCR+CSR+c- Jun immune cells.
[0213] At various points during preparation of a composition, it can be necessary or beneficial to cryopreserve a cell. The terms "frozen/freezing" and "cryopreserved/cryopreserving" can be used interchangeably. Freezing includes freeze drying.
[0214] As is understood by one of ordinary skill in the art, the freezing of cells can be destructive (see Mazur, P., 1977, Cryobiology 14:251 -272) but there are numerous procedures available to prevent such damage. For example, damage can be avoided by (a) use of a cryoprotective agent, (b) control of the freezing rate, and/or (c) storage at a temperature sufficiently low to minimize degradative reactions. Exemplary cryoprotective agents include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183: 1394- 1395; Ashwood- Smith, 1961 , Nature 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, 1960, Ann. N.Y. Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21 : 157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan and Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954, Biochem. J. 56:265), and inorganic salts (Phan and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan and Bender, 1961 , in Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery ed., Butterworth, London, p. 59). In particular embodiments, DMSO can be used. Addition of plasma (e.g., to a concentration of 20-25%) can augment the protective effects of DMSO. After addition of DMSO, cells can be kept at 0° C until freezing, because DMSO concentrations of 1% can be toxic at temperatures above 4° C.
[0215] In the cryopreservation of cells, slow controlled cooling rates can be critical and different cryoprotective agents (Rapatz et al., 1968, Cryobiology 5(1 ): 18-25) and different cell types have different optimal cooling rates (see e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology 3(1 ): 12-18; Lewis, et al., 1967, Transfusion 7(1 ): 17-32; and Mazur, 1970, Science 168:939- 949 for effects of cooling velocity on survival of stem cells and on their transplantation potential). The heat of fusion phase where water turns to ice should be minimal. The cooling procedure can be carried out by use of, e.g, a programmable freezing device or a methanol bath procedure. Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling.
[0216] In particular embodiments, DMSO-treated cells can be pre-cooled on ice and transferred to a tray containing chilled methanol which is placed, in turn, in a mechanical refrigerator (e.g., Harris or Revco) at -80° C. Thermocouple measurements of the methanol bath and the samples indicate a cooling rate of 1° to 3°C/minute can be preferred. After at least two hours, the specimens can have reached a temperature of - 80° C and can be placed directly into liquid nitrogen (-196° C).
[0217] After thorough freezing, the cells can be rapidly transferred to a long-term cryogenic storage vessel. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196° C) or vapor (-1° C). Such storage is facilitated by the availability of highly efficient liquid nitrogen refrigerators.
[0218] Further considerations and procedures for the manipulation, cryopreservation, and long-term storage of cells, can be found in the following exemplary references: U.S. Patent Nos. 4,199,022; 3,753,357; and 4,559,298; Gorin, 1986, Clinics In Haematology 15(1 ): 19- 48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107- 186; Livesey and Linner, 1987, Nature 327:255; Linner et al., 1986, J. Histochem. Cytochem. 34(9): 1 123-1 135; Simione, 1992, J. Parenter. Sci. Technol. 46(6):226-32).
[0219] Following cryopreservation, frozen cells can be thawed for use in accordance with methods known to those of ordinary skill in the art. Frozen cells are preferably thawed quickly and chilled immediately upon thawing. In particular embodiments, the vial containing the frozen cells can be immersed up to its neck in a warm water bath; gentle rotation will ensure mixing of the cell suspension as it thaws and increase heat transfer from the warm water to the internal ice mass. As soon as the ice has completely melted, the vial can be immediately placed on ice.
[0220] In particular embodiments, methods can be used to prevent cellular clumping during thawing. Exemplary methods include: the addition before and/or after freezing of DNase (Spitzer et al., 1980, Cancer 45:3075-3085), low molecular weight dextran and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20: 17-24), etc. As is understood by one of ordinary skill in the art, if a cryoprotective agent that is toxic to humans is used, it should be removed prior to therapeutic use. DMSO has no serious toxicity.
[0221] Exemplary carriers and modes of administration of cells are described at pages 14- 15 of U.S. Patent Publication No. 2010/0183564. Additional pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).
[0222] In particular embodiments, cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically-effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.
[0223] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, a carrier for infusion includes buffered saline with 5% HAS or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
[0224] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
[0225] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2- phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha- monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (z.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.
[0226] Where necessary or beneficial, compositions can include a local anesthetic such as lidocaine to ease pain at a site of injection.
[0227] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3 -pentanol.
[0228] Therapeutically effective amounts of cells within compositions can be greater than 102 cells, greater than 103 cells, greater than 104 cells, greater than 105 cells, greater than 106 cells, greater than 107 cells, greater than 108 cells, greater than 109 cells, greater than IO10 cells, or greater than 1011 cells.
[0229] In compositions and formulations disclosed herein, cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater than 104 cells/ml, 107 cells/ml or 108 cells/ml.
[0230] Also provided herein are nucleic acid compositions (such as pharmaceutical compositions, also referred to herein as formulations) comprising any of the nucleic acids encoding the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun polypeptides described herein. In some embodiments, the nucleic acid composition is a pharmaceutical composition. In some embodiments, the nucleic acid composition further comprises any of an isotonizing agent, an excipient, a diluent, a thickener, a stabilizer, a buffer, and/or a preservative; and/or an aqueous vehicle, such as purified water, an aqueous sugar solution, a buffer solution, physiological saline, an aqueous polymer solution, or RNase free water. The amounts of such additives and aqueous vehicles to be added can be suitably selected according to the form of use of the nucleic acid composition.
[0231] The compositions and formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage. The compositions and formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous injection.
[0232] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
[0233] In some embodiments, engineered immune cells can be harvested from a culture medium, and washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline (e.g., phosphate buffered saline), physiological saline, water, Hanks' solution, Ringer’s solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. It is preferred that the carrier is isotonic. In some embodiments, the carrier can be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol may also be included.
[0234] The pharmaceutical immune cell compositions (such as pharmaceutical T cell compositions) may be administered in a therapeutically effective amount to a cancer patient systemically (e.g., through intravenous or portal vein injection) or locally (e.g., through intratumoral injection). In some embodiments, the compositions such as those targeting GPC3 are used to treat a patient with hepatocellular carcinoma (HCC), stomach cancer, pancreatic cancer, or a cancer in the reproductive system (see, e.g., Wang and Wang, Canadian J Gastroent Hep. (2018) art. 9049252). As used herein, the term “treatment” or “treating” refers to an approach for obtaining beneficial or desired results in the treated subject. Such results include, but are not limited to: alleviating one or more symptoms resulting from the disease (e.g., HCC), diminishing the extent of the disease (e.g, reducing tumor volumes), stabilizing the disease (e.g, preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence or relapse of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, improving the quality of life, restoring body weight, and/or extension of survival (e.g., overall survival or progression-free survival).
[0235] A therapeutically effective amount of the composition refers to the number of engineered immune cells (such as T cells) sufficient to achieve a desired clinical endpoint. In some embodiments, a therapeutically effective amount contains more than 104, 105, 106, 107, 108, or 109 of the engineered cells.
[0236] The pharmaceutical composition in some embodiments comprises the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
[0237] In certain embodiments, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.
[0238] The cells and compositions in some embodiments are administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration can be autologous or allogeneic. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present disclosure (e.g., a pharmaceutical composition containing a genetically modified cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
[0239] In one aspect, the present disclosure provides pharmaceutical compositions comprising the nucleic acid molecules for expressing the anti-GPC3 caTCR, the anti-GPC3 CSR, and c-Jun. The nucleic acid molecules may be as described above, such as the viral vectors (e.g., lentiviral vectors) described above. The pharmaceutical compositions may be used ex vivo to engineer T or progenitor cells, which are then introduced to the patient. The pharmaceutical compositions comprise the nucleic acid molecules or the recombinant viruses whose genome comprises the expression cassettes for the anti-GPC3 caTCR, the anti-GPC3 CSR, and c-Jun and a pharmaceutically acceptable carrier such as a buffered solution that optionally comprises other agents such as preservatives, stabilizing agents, and the like.
[0240] The pharmaceutical compositions may be provided as articles of manufacture, such as kits, that include vials (e.g., single-dose vials) comprising the biological materials (the cells or the nucleic acid molecules or recombinant viruses) and optionally instructions for use.
V. Methods of Treatment
[0241] The expression constructs and/or compositions (such as pharmaceutical compositions) of the invention can be administered to individuals (e.g., mammals such as humans) to treat a disease and/or disorder involving abnormally high GPC3 expression (also referred to herein as a “GPC3 -positive” disease or disorder), including, for example, cancer (such as hepatocellular carcinoma (HCC)).
[0242] Thus, in some embodiments, the present application provides a method of treating a GPC3-positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide. In some embodiments, the anti-GPC3 caTCR is any of the anti- GPC3 caTCRs provided herein. In some embodiments, the anti-GPC3 CSR is any of the anti- GPC3 CSRs provided herein. In some embodiments, the c-Jun polypeptide is any of the c-Jun polypeptides described herein.
[0243] In some embodiments, provided herein is a method of treating a cancer in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide, such as any of the anti-GPC3 caTCRs, anti-GPC3 CSRs, and c-Jun polypeptides described herein. In some embodiments, the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma. In some embodiments, the cancer is HCC. In some embodiments, the cancer is HCC and the treating comprises preventing the spread of the cancer, e.g., inhibiting (such as preventing) metastasis of the cancer. In some embodiments, the cancer is metastatic HCC. In some embodiments, the individual is human. [0244] In some embodiments, provided herein is a method of treating a GPC3 -positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligandbinding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligand-binding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the TCRM of the anti-GPC3 caTCR is derived from a human y/8 TCR. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30). In some embodiments, the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from CD30 (e.g., human CD30). In some embodiments, the human c- Jun polypeptide is a wildtype human c-Jun polypeptide. In some embodiments, the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
[0245] In some embodiments, provided herein is a method of treating a GPC3 -positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module comprising: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC- CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligandbinding module comprising: (a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC- CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligand-binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3 caTCR comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the ligand binding module of the anti-GPC3 CSR comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises transmembrane domains derived from CD30 (e.g., human CD30). In some embodiments, the co-stimulatory immune cell signaling module of the anti- GPC3 CSR is derived from CD30 (e.g., human CD30). In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti- GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the human c-Jun polypeptide is wildtype human c-Jun. In some embodiments, the human c- Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
[0246] In some embodiments, provided herein is a method of treating a GPC3 -positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing: one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigenbinding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand-binding module comprising a scFv that is capable of binding or interacting with GPC3; ii) a transmembrane module derived from CD30; and iii) a costimulatory immune cell signaling module derived from CD30 that is capable of providing a co-stimulatory signal to the immune cell, wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the Fab comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-GPC3 CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
[0247] In some embodiments, provided herein is a method of treating a GPC3 -positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (such as a pharmaceutical composition) comprising one or more immune cells expressing a polycistronic expression construct comprising an expression cassette for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) an anti-GPC3 CSR comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence for SEQ ID NO: 1, coding sequences for SEQ ID NOs: 28 and 29, and a coding sequence for SEQ ID NO: 33. In some embodiments, coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF-la promoter. In some embodiments, the construct is a viral vector (such as a lentiviral vector). In some embodiments, the cancer is selected, for example, from the group consisting of HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
[0248] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
EXAMPLES
[0249] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
[0250] In the studies described in Examples 1-3 below, the cell lines HepG2-luc (Caliper, derived from ATCC HB-8065) and Hep3B (ATCC HB-8064) were obtained from Caliper and the American Type Culture Collection. Both HepG2 and Hep3B are hepatocellular carcinoma (HCC) cell lines that express glypican-3 (GPC3). The cell lines were cultured in RPMI-1640 (Gibco) or DMEM (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C/5% CO2.
[0251] Two lentiviral vectors were constructed. The first one, named “GPC3-caTCR + GPC3-CD30-CSR” or “Construct 1” herein, contained a tri-cistronic expression cassette encoding the GPC3-specific caTCR (SEQ ID NO: 30 and 31) and the GPC3-specific CSR comprising a co-stimulatory domain derived from CD30, i.e., GPC3-CD30-CSR (SEQ ID NO: 16). The second vector, named “c-Jun + GPC3-caTCR + GPC3-CD30-CSR” or “Construct 2” herein, contained a quad-cistronic expression cassette encoding the GPC3- specific caTCR (SEQ ID NO: 30 and 31), the GPC3-CD30-CSR (SEQ ID NO: 16), and human c-Jun (SEQ ID NO: 1). In each vector, the expression cassette was under the transcriptional control of an EF- la promoter and the coding sequences for the different polypeptides were linked in frame by coding sequences for self-cleaving peptides (F2A, T2A or P2A, as illustrated in FIG. 1A). The recombinant viruses were produced by transfection of 293T cells with vectors encoding the constructs using known lentiviral production protocols and packaging systems.
[0252] Primary human T cells were used for lentiviral transduction after one-day stimulation with CD3/CD28 beads (Dynabeads®, Invitrogen) in the presence of interleukin-2 (IL-2) at 100 U/mL. Concentrated lentiviruses were applied to the T cells in 6-well plates coated with Retronectin® (Takara) and incubated for 96 hours. Transduction efficiencies were assessed by flow cytometry on day 9, using a PE-conjugated anti-human F(ab’)2 antibody. Flow cytometry data were collected using a BD FACSCanto II™ and analyzed using the FlowJo software package.
[0253] For in vivo studies, the anti-tumor activity of primary human T cells expressing Construct 1 or Construct 2 were tested in two liver cancer models, namely HepG2-luc (Studies 1 and 2) and Hep3B models (Study 3). Luciferase-expressing HepG2 (HepG2-luc) cells or Hep3B cells were implanted subcutaneously (s.c.) in the right flank of NSG-MHC I/II DKO mice (described in FIG. IB). When tumors reached about 150 mm3 in size (20 days after the tumor cell implantation), the mice were intravenously (i.v.) injected with the modified T cells.
[0254] Health effects resulting from the T cell infusions in mice were assessed by monitoring the animals’ general appearance, body weight, and other clinical signs of adverse response (including hypothermia, labored respiration, and hind-limb paralysis/weakness). In addition, peripheral blood T cell count was measured on day 14 after T cell dosing.
Example 1: Characterization of anti-GPC3 caTCR + anti-GPC3 CSR T cells overexpressing c-Jun in in vitro long-term target engagement assays
[0255] This Example describes the effects of c-Jun overexpression on the cytotoxicity of human T cells that were engineered to express an anti-GPC3 caTCR and an anti-GPC3 CSR comprising a co-stimulatory domain derived from CD30 (z.e., GPC3-CD30-CSR). In particular, this Example shows that c-Jun overexpression promotes long-term target-cell- killing capabilities in T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR. T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR, with or without c-Jun overexpression, perform comparably regarding long term T cell exhaustion.
[0256] Primary T cells isolated from healthy human donors were transduced with lentiviral Construct 1 (z.e., GPC3-caTCR + GPC3-CD30-CSR) or Construct 2 (z.e., c-Jun + GPC3- caTCR + GPC3-CD30-CSR) for 7-9 days. Tumor cytotoxicity was assayed by a Cytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. CD3+ T cells were prepared from PBMC-enriched whole blood using an EasySep™ Human T Cell Isolation Kit (StemCell Technologies), which negatively depletes cells expressing CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A. Human T cells were activated and expanded with CD3/CD28 Dynabeads® (Invitrogen) according to the manufacturer’s protocol. Activated T cells (ATCs) were cultured and maintained in RPMI- 1640 medium with 10% FBS plus 100 U/mL IL-2 and used at day 7-14. The effector cells were normalized by caTCR expression (caTCR+) based on human F(ab’)2 staining (adjusting all effector cell samples to the same percentage of receptor+ (caTCR+) cells among the total number of T cells using un-transduced/mock-transduced T cells).
[0257] T cells and target cells were co-cultured with starting effector-to-target ratio (E:T ratio) of 1 : 1. Specifically, 50,000 caTCR+ T cells and 50,000 HepG2 cells were incubated together in each well in RPML1640 and 10% FBS with no cytokines. To assess long-term killing potential of the T cells, the cells were re-challenged with 100,000 HepG2 cells per well every 7 days. The numbers of remaining target cells and caTCR+ T cells were quantified after each target cell engagement.
[0258] The comparison of the long-term killing (represented by quantities of remaining target cells and remaining T cell numbers) are shown in Table 3. T cells expressing Construct 2 cleared target cells through more re-challenges than T cells expressing Construct 1, indicating that c-Jun promotes long-term target-cell-killing capabilities in anti-GPC3 caTCR+ anti-GPC3 CSR+ T cells.
[0259] Subpopulations of CD4 or CD8 single positive caTCR+ T cells were analyzed by flow cytometry after each target cell engagement. The data in Table 3 indicates that c-Jun overexpression resulted in higher percentages of CD4+ T cells but lower percentages of CD8+ T cells, suggesting that c-Jun expression promoted preferential expansion of CD4+ T cells. [0260] To determine whether transduction with either Construct 1 or 2 has an effect on T cell differentiation during the long-term killing assay, CD8+ Receptor+ cells were assayed by flow cytometry for expression of CCR7 and CD45RA. Naive T cells are characterized by CCR7+ and CD45RA+, whereas memory T cells are characterized by CCR7+, and CD45RA". As shown in Table 3, the expression levels of CCR7 and the number of memory and naive T cells are comparable between T cells expressing Construct 1 and those expressing Construct 2. These results indicate that c-Jun does not affect the differentiation of anti-GPC3 caTCR+ anti-GPC3 CSR+ memory T cells.
[0261] To examine the levels of exhaustion markers expressed on T cells transduced with either Construct 1 or Construct 2 during the long-term killing assay, CD3+ T cells were prepared and activated with CD3/CD28 Dynabeads® as described above. The T cells were transduced with lentiviral Construct 1 or Construct 2 for 7-9 days. The effector cells were normalized by caTCR+ based on human F(ab’)2 staining. The T cells were incubated and rechallenged with HepG2 cells, as described above. The expression levels of exhaustion markers PD-1, LAG-3, and TIM-3 (BioLegend®) on the caTCR+CD8+ T cells and caTCR+CD4+ T cells were analyzed by flow cytometry on selected days after each target cell engagement. PD-1, LAG-3, and TIM-3 are inhibitory receptors that accumulate on T cells as T cells lose function; thus, these molecules are used as markers for T cell exhaustion.
[0262] As shown in Table 3, the two groups of T cells displayed similar levels of the exhaustion markers. These results demonstrate that anti-GPC3 caTCR+ anti-GPC3 CSR+ T cells with or without c-Jun overexpression perform comparably regarding long term exhaustion.
Table 3. In vitro long-term target engagement results
Figure imgf000100_0001
Figure imgf000101_0001
Example 2: In vivo efficacy of anti-GPC3 caTCR + anti-GPC3 CSR T cells overexpressing c-Jun in HepG2-luc mouse model re-challenge study
[0263] This Example shows that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR with c-Jun overexpression increased tumor-killing in vivo compared with T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.
[0264] Primary human T cells from two different donors expressing Construct 1 or 2 were assessed for their in vivo cytotoxic potency in HepG2-luc model, as described above. In these studies, the mice were injected i.v. with one of the following: (1) 5* 106 un-transduced donor- matched (Mock) primary human T cells, (2) 5* 106 primary human T cells expressing Construct 1, (3) 2* 106 primary human T cells expressing Construct 1, (4) 5* 106 primary human T cells expressing Construct 2, or (5) 2* 106 primary human T cells expressing Construct 2 (n=10 mice/group). To simulate tumor relapse, mice in all treatment groups were subjected to tumor re-challenge studies, wherein HepG2-luc cells were implanted s.c. in the left flank around day 70 post T cell dosing.
[0265] Around day 10 post-injection of T cells in all four treatment groups (z.e., high dose of Construct 1, low dose of Construct 1, high dose of Construct 2, low dose of Construct 2), tumors started to regress; complete tumor regression was achieved around 20 days (FIG. 2). No significant difference was observed between the primary tumor curves of animals treated with T cells expressing Construct 1 and Construct 2. These results show that c-Jun overexpression does not affect the primary tumor-killing potential of the “GPC3-caTCR + GPC3-CD30-CSR” T cells.
[0266] Interestingly, an enhancement effect of c-Jun was observed when the animals were subjected to tumor re-challenge. T cells expressing Construct 2 performed significantly better than T cells expressing Construct 1, indicating c-Jun boosted the tumor-killing potential of the “GPC3-caTCR + GPC3-CD30-CSR” T cells in the tumor re-challenge study (FIG. 3). Additionally, in one study, mice injected with the higher dose of T cells expressing Construct 2 had higher peripheral blood T cell count (FIGs. 4A and 4B). Example 3: In vivo efficacy of anti-GPC3 + anti-GPC3 CSR T cells overexpressing c- Jun in Hep3B mouse model
[0267] This Example shows that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR with c-Jun overexpression have increased T cell expansion in vivo compared with T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.
[0268] Primary human T cells expressing Construct 1 or 2 were assessed for their in vivo cytotoxic potency in Hep3B model. The mice were injected i.v. with one of the following: (1) 2.5* 106 un-transduced donor-matched (Mock) primary human T cells, (2) 2.5* 106 primary human T cells expressing Construct 1, (3) 1.5>< 106 primary human T cells expressing Construct 1, (4) 2.5>< 106 primary human T cells expressing Construct 2, or (5) 1.5>< 106 primary human T cells expressing Construct 2 (n=10 mice/group). As shown in FIG. 5, tumor regrowth was observed in all groups after initial regression. T cells expressing Construct 2 repressed the size of relapsing tumors to a higher extent than those expressing Construct 1. At the higher dose of 2.5M, the difference between the tumor repressing effects of Construct 1 and 2 as measured by tumor volume was less discernable. In addition, mice in the Construct 2 groups in both doses had higher numbers of peripheral blood T cells, indicating c-Jun positively affects anti-GPC3 caTCR+ anti-GPC3 CSR+ T cell expansion in vivo (FIG. 4C)
Example 4: Characterization of T cells expressing various anti-GPC3 caTCRs + anti- GPC3 CSRs in in vitro long-term target engagement assays
[0269] This Example describes the cytotoxicity of human T cells that are engineered to express various anti-GPC3 caTCRs (e.g., GPC3-caTCR) comprising different sequences, and various anti-GPC3 CSRs comprising different sequences and a co-stimulatory domain derived from human CD30 (z.e., GPC3-CD30-CSR), in addition to a human c-Jun polypeptide.
[0270] Primary T cells isolated from healthy human donors are transduced with one of the sets of expression constructs described in Table 4 below.
Table 4. Exemplary Expression Constructs Comprising Different Combinations of Anti-GPC3 Moieties
Figure imgf000102_0001
Figure imgf000103_0001
[0271] T cells expressing each group of expression constructs are co-cultured with HepG2 cells, and assayed for cytotoxicity and exhaustion, as described in Example 1 above.
[0272] Construct c-Jun+GPC3-3 caTCR+GPC3-2-CD30 CSR (Table 4) shows the highest long-term target-cell-killing capabilities and lowest exhaustion, compared to the other constructs described in Table 4.
Example 5: Characterization of T cells expressing anti-GPC3 caTCRwith different anti-GPC3 CSRs
[0273] This Example characterizes human T cells that are engineered to express an anti- GPC3 caTCR (e.g, GPC3-caTCR) in combination with various anti-GPC3 CSRs (e.g., GPC3-CSR) comprising different co-stimulatory fragments (e.g., co-stimulatory fragments comprising a co-stimulatory domain and a transmembrane domain), in addition to a human c- Jun polypeptide.
[0274] Primary T cells isolated from healthy human donors are transduced with the one of the sets of expression constructs described in Table 5 below. Each exemplary GPC3 -caTCR encodes the VH/VL sequences of GPC3-3 (see, Sequence listing), and each exemplary GPC3- CSR encodes the VH/VL sequences of GPC3-2 (see, Sequence listing). Table 5. Exemplary expression constructs comprising anti-GPC3-CSRs comprising different co-stimulatory fragments
Figure imgf000104_0001
[0275] Assays comparing T cells expressing the constructs described in Table 5 (e.g., T cells expressing the same anti-GPC3-caTCR e.g., GPC3-3 caTCR) and c-Jun, but with different anti-GPC3-CSR comprising different co-stimulatory fragments) are performed and described in Examples 5A-5G.
[0276] Overall, Examples 5A-5G show that c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM- CD30 IC-CSR and c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR perform equally well, and better than the other anti-GPC3-CSRs.
Example 5 A: Short-term in vitro cancer cell killing assay
[0277] T cells expressing each of the constructs described in Table 5 are co-cultured with HepG2 cells and tumor cytotoxicity was assayed by a Cytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH) released into culture supernatants, as described in Example 1.
[0278] T cells expressing either c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM-CD30 IC-CSR or c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR have higher killing efficacies than the other constructs outlined in Table 5, without a CSR or CSR comprising other costimulatory fragments.
[0279] The short-term killing ability of the various T cells expressing the constructs of Table 5 is also determined by measuring the amounts/levels of cytokines released from T cells upon engagement with HepG2 cells. The levels of cytokine release in the supernatant after 16 hour co-culture are quantified with Luminex Magpix technology using BioRad Bio- Plex kits or by ELISA. T cells with high cytotoxic potency secrete high levels of cytokines that are related to T cell activity, such as TNFa, GM-CSF, IFNy, and IL-2.
[0280] T cells expressing a c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have higher killing efficacies than those without any CSR, and higher than or about the same killing efficacies as those with CSRs that do not comprise a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains (see, Table 5).
Example 5B: Proliferation potential and persistence assays
[0281] The proliferation and persistence of genetically modified T cells is crucial for the success of adoptive T-cell transfer therapies when treating cancers. To assay the effect of particular CSRs on T-cell proliferation and persistence, T cells expressing each of the constructs of Table 5 are labeled with the intracellular dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and the dilution of the dye is observed as the T cells divide when stimulated with tumor cells. Persistence of the T cells is measured by counting the number of CFSE-positive cells remaining at an indicated day.
[0282] T cells expressing each of the constructs shown in Table 5 are serum starved overnight and labeled with CFSE using CellTrace CFSE (Thermo Fisher). 50,000 to 100,000 T cells are incubated at an effector cell to target cell ratio (E:T ratio) of 2: 1, and flow cytometry is used to observe serial dilution of the CFSE dye as the T cells divide at an indicated day. The total number of T cells are counted with FACS.
[0283] T cells expressing a c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain proliferate more than corresponding CAR T cells without any CSR, and proliferate more than or about the same as corresponding CAR T cells expressing CSRs that do not comprise a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains. Example 5C: In vitro T cell and tumor cell counts after multi-week engagements [0284] A FACS based assay for counting target cells is used to compare the long-term killing potential of T cells expressing each of the constructs described in Table 5. T cells expressing each of the constructs described in Table 5 show comparable survival post target cell engagement.
[0285] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain persist for a longer period of time over multiple engagements of tumor cells and kill more tumor cells than corresponding CAR T cells without CSR, and about the same as CAR T cells with CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains, if not better.
[0286] A FACS based assay for counting T cells and target cells is used to compare the long-term survival and target-cell killing potential of T cells expressing the constructs described in Table 5. The T cells are co-cultured and re-challenged with target cells as described in Example 1. The numbers of remaining target cells and total T cells are quantified with FACS on various days after each target cell engagement.
[0287] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain persist/survive for a longer period of time over multiple engagements of tumor target cells and kill more tumor cells than corresponding T cells without any CSR, and survive better and/or kill more tumor cells than or about the same as corresponding T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains.
Example 5D: In vitro T cell exhaustion
[0288] To examine the levels of exhaustion markers expressed on T cells transduced with the constructs described in Table 5, the expression levels of exhaustion markers TIGIT, PD-1, LAG-3, and TIM-3 (BioLegend®) on the caTCR+CD8+ T cells and caTCR+CD4+ T cells are analyzed by flow cytometry on selected days after each target cell engagement, as described in Example 1.
[0289] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have decreased long term exhaustion markers than corresponding T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains. Example 5E: In vivo cytokine release
[0290] Primary human T cells expressing each of the constructs of Table 5 are assessed for their in vivo cytotoxic potency in a HepG2-luc and a Hep3B mouse model, as described in Examples 2 and 3, respectively. Briefly, to determine the level of cytokine release in vivo, key cytokines, including those related to clinical cytokine release syndrome, are analyzed 16, 24, 48, and 72 hours after the T cells are administered. Cytokine levels are quantified with Luminex Magpix technology using BioRad Bio-Plex kits.
[0291] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain secrete higher levels of cytokines that are related to T cell activity, such as TNFa, GM-CSF, IFNy, and IL-2, than corresponding T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
Example 5F: Differentiation of T-cell subsets over time (CCR7/CD45RA) and memory T cell quantification
[0292] Proliferation and survival of T cells expressing the constructs described in Table 5 is measured before and after target cell engagement in two independent flow cytometric assays.
[0293] FACS analysis of T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain show a greater level of expression of the T cell differentiation markers CCR7 and CD45RA , as well as increased percentage of memory and naive T cells compared to T cells without any CSR or T cells expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains, prior to target engagement.
[0294] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3-CD30-CSR develop into and maintain a high memory T cell population after target stimulation, including central memory and effector memory T cells. To determine the effect of different CSRs on T cells’ ability to develop into and maintain memory T cells, the cell surface expression of memory T cell markers CCR7 and CD45RA is measured. As known in the field, T cells with high CCR7 expression levels and low CD45RA expression levels are considered central memory T cells, T cells with low CCR7 and low CD45RA expression levels are considered effector memory T cells, T cells with low CCR7 and high CD45RA expression levels are considered effector T cells, while T cells with high CCR7 and high CD45RA are naive T cells which are the initial type of T cells before target/antigen challenge/recognition (Mahnke et al., Eur J Immunol. 43(11):2797-809, 2013). In response to an antigen encounter, naive T cells proliferate and differentiate into effector cells, most of which carry out the job of destroying targets and then die, while a small pool of T cells ultimately develops into long- lived memory T cells which can store the T cell immunity against the specific target. Among the memory T cells, the central memory T cells are found to have longer lives than effector memory T cells and are capable of generating effector memory T cells, but not vice versa. Therefore, the ability to develop into and maintain memory T cells, especially central memory T cells, is an important and desired feature for potentially successful T cell therapies. [0295] T cells expressing c-Jun and an anti-GPC3 caTCR but no CSR are incubated with HepG2 cells at an E:T ratio of 2: 1 (e.g., 100,000 receptor+ T cells and 50,000 HepG2 cells in each well on a 96- well plate) for 7 days. The cells are then re-challenged with 50,000- 100,000 HepG2 cells per well every 7 days. The T cells in the other groups expressing different CSRs are incubated with target cells at an E:T ratio of 1 :2 (e.g., 25,000 receptor+ T cells and 50,000 HepG2 cells in each well) for 7 days. The cells are then re-challenged with 50,000-100,000 HepG2 cells per well every 7 days.
[0296] In some experiments, the T cell and HepG2 cell mixtures are diluted 1 :6 before the fourth and fifth HepG2 cell engagement, to avoid the overcrowding of T cells due to the significant T cell expansion, so that only one sixth of the previously remaining cells are rechallenged with 50,000-100,000 HepG2 cells.
[0297] On selected days after each HepG2 cell engagement, the entire cell mixture in a well from each sample is stained with antibodies against CCR7 and CD45RA and analyzed by flow cytometry. Receptor+ T cell numbers are counted, and cells are grouped into various T cell types based on their CCR7 and CD45RA expression levels: central memory T cells (CD45RA'CCR7+), effector memory T cells (CD45RA'CCR7'), effector T cells (CD45RA+ CCR7" ), and naive T cells (CD45RA+CCR7+). Percentages of various types of T cells among the total number of receptor+ T cells are calculated. In some experiments, the cells are also stained with antibodies against CD8 or CD4 to determine the CD8 (cytotoxic T cells) and CD4 (helper T cells) composition of the T cell types.
[0298] Proliferation and survival of T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR are measured before and after target cell engagement. T cells expressing c- Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain are able to develop into and maintain high numbers and high percentages of central memory T cells upon engagement with HepG2 cells, higher than those without any CSR or those expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4-1BB IC domains.
Example 5G: In Vivo efficacy of T cells in a HepG2 or a Hep3B mouse model [0299] About 107 HepG2 tumor cells or Hep3B are implanted subcutaneously in NSG mice and allowed to form a solid tumor mass 150mm3. 5xl06 T cells expressing each of the constructs described in Table 5 are injected i.v. into the tumor bearing mice. 3 weeks after T- cell dosing, the mice are sacrificed and tumors removed, fixed, and sectioned onto slides. Tumor sections are stained with CD3 antibody to visualize the T cells that are present within the solid tumor. Quantification of the number of CD3+ cells can be used to score the tumor infiltration ability of the T cells (T-cell/mm2).
[0300] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain have higher in vivo tumor infiltration/penetration rates/levels (z.e., higher numbers of T cells/mm2) than those without any CSR or those expressing CSRs that do not have a CD30 IC domain but have a different co-stimulatory molecule’s IC domain, e.g., CD28 or 4- IBB IC domains.
LIST OF SEQUENCES
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Claims

1. One or more expression constructs comprising one or more expression cassettes for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR- TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to the immune cell, wherein the ligand-binding module and the costimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide.
2. The expression construct(s) of claim 1, wherein the c-Jun is a wildtype human c-Jun.
3. The expression construct(s) of claim 2, wherein the wildtype human c-Jun comprises at least about 90% identity to the amino acid sequence of SEQ ID NO: 1.
4. The expression construct(s) of claim 1, wherein the c-Jun is a mutant human c-Jun.
5. The expression construct(s) of claim 4, wherein the mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain.
6. The expression construct(s) of claim 4 or 5, wherein the mutant human c-Jun comprises:
(i) S63A and S73A substitutions as compared to SEQ ID NO: 1; or, (ii) a deletion between amino acid residues 2 and 102 or between amino acid residues 30 and 50 as compared to SEQ ID NO: 1.
7. The expression construct(s) of any one of claims 1-6, wherein the GPC3 is a cellsurface bound GPC3.
8. The expression construct(s) of any one of claims 1-7, wherein the TCRM is derived from a human y/8 TCR.
9. The expression construct(s) of any one of claims 1-8, wherein the caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the caTCR.
10. The expression construct(s) of claim 9, wherein the stabilization module is selected from the group consisting of a TCR constant domain, a CH1-CL module, a CH2-CH2 module, a CH3- CH3 module, and a CH4-CH4 module.
11. The expression construct(s) of claim 10, wherein the stabilization module is derived from a human protein.
12. The expression construct(s) of claim 10 or 11, wherein the CL contained in the CH1-CL module is derived from a kappa antibody light chain or lambda antibody light chain.
13. The expression construct(s) of any one of claims 1-12, wherein the antigen-binding module of the caTCR is a Fab, a Fab', a (Fab')2, an Fv, or a single chain Fv (scFv).
14. The expression construct(s) of any one of claims 1-13, wherein the antigen-binding module of the caTCR comprises:
(i) a heavy chain variable domain (VH) comprising a heavy chain complementaritydetermining region (HC-CDR) 1 (HC-CDR1) comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable domain (VL) comprising a light chain complementarity determining region (LC-CDR) 1 (LC-CDR1) comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions;
(ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; or,
(iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 3 amino acid substitutions.
15. The expression construct(s) of claim 14, wherein the antigen-binding module of the caTCR comprises:
(i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11;
(ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or,
(iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
16. The expression construct(s) of any one of claims 1-15, wherein the antigen-binding module of the caTCR comprises:
(i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25;
(ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or,
(iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 29.
17. The expression construct^ s) of claim 16, wherein the antigen-binding module of caTCR comprises:
(i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25;
(ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or,
(iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
18. The expression construct(s) of any one of claims 1-17, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the first TCRD and a second polypeptide chain comprising the second TCRD.
19. The expression construct(s) of any one of claims 1-18, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR 5 chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR y chain.
20. The expression construct(s) of any one of claims 1-18, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising a VH fused to a TCRD derived from a TCR y chain and a second polypeptide chain comprising a VL fused to a TCRD derived from a TCR 5 chain.
21. The expression construct(s) of any one of claims 1-19, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 31.
22. The expression construct(s) of any one of claims 1-18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 10, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 5.
23. The expression construct^ s) of any one of claims 1-19, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 63, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 64.
24. The expression construct(s) of any one of claims 1-18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 65, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 66.
25. The expression construct(s) of any one of claims 1-19, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 67, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 68.
26. The expression construct(s) of any one of claims 1-18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 69, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 70.
27. The expression construct^ s) of any one of claims 1-26, wherein the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30, CD28, CD3s, CD3< CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
28. The expression construct(s) of any one of claims 1-27, wherein the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.
29. The expression construct(s) of any one of claims 1-28, wherein the co-stimulatory immune cell signaling module is derived from the intracellular domain of a co-stimulatory receptor of a TCR.
30. The expression construct(s) of claim 29, wherein the co-stimulatory receptor is selected from the group consisting of CD30, CD28, 4-IBB, 0X40, ICOS, CD27, and CD40.
31. The expression construct(s) of any one of claims 1-30, wherein the co-stimulatory immune cell signaling module of the CSR is derived from human CD30.
32. The expression construct(s) of claim 31, wherein the co-stimulatory immune cell signaling module of human CD30 comprises the amino acid sequence of SEQ ID NO: 44.
33. The expression construct(s) of any one of claims 1-32, wherein the co-stimulatory immune cell signaling module and the transmembrane domain of the CSR are both derived from human CD30.
34. The expression construct(s) of any one of claims 1-33, wherein the ligand-binding module of the CSR comprises:
(i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to about 3 amino acid substitutions, an LC- CDR2 comprising the amino acid sequence of GDN, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 3 amino acid substitutions;
(ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of YDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a variant thereof comprising up to about 3 amino acid substitutions; or,
(iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 3 amino acid substitutions; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of DDS, or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or a variant thereof comprising up to about 3 amino acid substitutions.
35. The expression construct(s) of claim 34, wherein the ligand-binding module of the CSR comprises:
(i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising the amino acid sequence of GDN, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or,
(iii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23.
36. The expression construct(s) of any one of claims 1-35, wherein the ligand-binding module of the CSR comprises:
(i) a VH comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 24, and a VL comprising the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 25;
(ii) a VH comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 26, and a VL comprising the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 27; or,
(iii) a VH comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 28, and a VL comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 29.
37. The expression construct(s) of claim 36, wherein the ligand-binding module of the CSR comprises:
(i) a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or,
(iii) a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
38. The expression construct(s) of any one of claims 1-37, wherein the ligand-binding module of the CSR comprises:
(i) SEQ ID NO: 32, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 32;
(ii) SEQ ID NO: 33, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 33; or,
(iii) SEQ ID NO: 34, or an amino acid sequence comprising at least about 90% sequence identity to SEQ ID NO: 34.
39. The expression construct(s) of any one of claims 1-38, wherein:
(i) the antigen-binding module of the caTCR comprises:
(a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:
19, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; and
(b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of DDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and
(ii) the ligand-binding module of the CSR comprises:
(a) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and
(b) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising the amino acid sequence of YDS, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
40. The expression construct(s) of any one of claims 1-39, wherein the antigen-binding module of the caTCR comprises a Fab that binds to GPC3, wherein the ligand-binding module of the CSR comprises a scFv that binds to GPC3, and wherein the transmembrane module and co-stimulatory immune cell signaling module of the CSR are both derived from CD30.
41. The expression construct(s) of claim 40, wherein the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and wherein:
(1) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27;
(2) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;
(3) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25;
(4) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27;
(5) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29;
(6) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; (7) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25;
(8) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or
(9) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29.
42. The expression construct(s) of claim 40 or 41, wherein the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and wherein:
(1) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33;
(2) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32;
(3) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34;
(4) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32;
(5) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33;
(6) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34; (7) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32;
(8) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; or
(9) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34.
43. The expression construct(s) of any one of claims 1-42, wherein the construct(s) are viral vectors.
44. The expression construct(s) of claim 43, wherein the viral vector(s) are selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.
45. The expression construct(s) of any one of claims 1-42, wherein the expression construct comprises a polycistronic expression cassette for expressing the caTCR, the CSR, and the c-Jun.
46. A polycistronic expression construct comprising an expression cassette for expressing: a) a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: i) an antigen-binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human y/8 TCR; b) a chimeric stimulating receptor (CSR) comprising: i) a ligand-binding module that is capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from an intracellular domain of human CD30; and c) a human c-Jun polypeptide.
47. The expression construct of claim 46, wherein the expression cassette comprises a coding sequence for SEQ ID NO: 1 and:
(i) coding sequences for SEQ ID NOs: 28 and 29 and a coding sequence for SEQ ID NO: 33;
(ii) coding sequences for SEQ ID NOs: 28 and 29 and a coding sequence for SEQ ID NO: 32;
(iii) coding sequences for SEQ ID NOs: 28 and 29 and a coding sequence for SEQ ID NO: 34;
(iv) coding sequences for SEQ ID NOs: 26 and 27 and a coding sequence for SEQ ID NO: 32;
(v) coding sequences for SEQ ID NOs: 26 and 27 and a coding sequence for SEQ ID NO: 33;
(vi) coding sequences for SEQ ID NOs: 26 and 27 and a coding sequence for SEQ ID NO: 34;
(vii) coding sequences for SEQ ID NOs: 24 and 25 and a coding sequence for SEQ ID NO: 32;
(viii) coding sequences for SEQ ID NOs: 24 and 25 and a coding sequence for SEQ ID NO: 33; or
(ix) coding sequences for SEQ ID NOs: 24 and 25 and a coding sequence for SEQ ID NO: 34.
48. The expression construct of claim 47, wherein coding sequences are separated in frame by a 2A-coding sequence or by an internal ribosomal entry site (IRES).
49. The expression construct of any one of claims 46-48, wherein the expression cassette comprises a constitutive or inducible promoter.
50. The expression construct of claim 49, wherein the promoter is an EF-la promoter.
51. The expression construct of any one of claims 46-50, wherein the construct is a viral vector.
52. The expression construct of claim 51, wherein the viral vector is selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia vectors, herpes simplex viral vectors, and Epstein-Barr viral vectors.
53. A recombinant virus comprising the polycistronic expression construct of any one of claims 50-52.
54. The recombinant virus of claim 53, wherein the polycistronic expression construct is a lentiviral vector.
55. A method of engineering immune cells, comprising:
(a) providing a starting cell population,
(b) introducing the expression construct(s) of any one of claims 1-52, or the recombinant virus of claim 53 or 54 into the starting cell population,
(c) optionally selecting cells that express the caTCR, the CSR, and the c-Jun, and
(d) deriving engineered immune cells from the cells of step (b) or (c).
56. The method of claim 55, wherein the immune cell is a T cell.
57. The method of claim 56, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer T cell, and a suppressor T cell.
58. The method of any one of claims 55-57, wherein the starting cell population comprises immune cells.
59. The method of any one of claims 55-58, wherein the starting cell population comprises autologous or allogeneic T cells.
60. The method of any one of claims 55-57, wherein the starting cell population comprises pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells.
61. A population of human cells comprising the expression construct(s) of any one of claims one of claims 1-52 or the recombinant virus of claim 53 or 54.
62. The population of human cells of claim 61, wherein the human cells are immune cells.
63. A population of immune cells obtained by the method of any one of claims 55-60.
64. The population of immune cells of claim 63, wherein the immune cells are human cells.
65. The immune cells of any one of claims 62-64, wherein the immune cells are T cells.
66. The immune cells of claim 65, wherein the T cells are CD8+ T cells.
67. The immune cells of any one of claims 62-66, wherein the immune cells express a lower level of an exhaustion marker compared to corresponding cells that do not overexpress c-Jun.
68. The immune cells of claim 67, wherein the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.
69. A pharmaceutical composition comprising the expression construct(s) of any one of claims 1-52, the recombinant virus of claim 53 or 54, or the cells of any one of claims 61-68, and a pharmaceutically acceptable carrier.
70. A method of killing target cells, comprising contacting the target cells with the pharmaceutical composition of claim 69 under conditions that allow killing of the target cells by the immune cells, wherein the target cells express the GPC3.
71. The method of claim 70, wherein the immune cells express a lower level of an exhaustion marker when in contact with the target cells, as compared to corresponding immune cells that do not comprise an exogenous nucleic acid molecule that causes c-Jun overexpression.
72. The method of claim 70 or 71, wherein the immune cells are T cells.
73. The method of claim 72, wherein the T cells are CD8+ T cells.
74. The method of claim 73, wherein the target cells are cancer cells.
75. The method of claim 74, wherein the co-stimulatory immune cell signaling module in the CSR is derived from human CD30 and the immune cells express a lower level of an exhaustion marker, as compared to corresponding immune cells engineered to express a CSR whose co-stimulatory immune cell signaling module is derived from human CD28.
76. The method of any one of claims 70-75, wherein the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.
77. A method of treating an individual in need thereof, comprising administering the pharmaceutical composition of claim 69 to the individual.
78. Use of the expression construct(s) of any one of claims 1-52, the recombinant virus of claim 53 or 54, or the pharmaceutical composition of claim 69, for the manufacture of a medicament for treating an individual in need thereof.
79. Use of the expression construct(s) of any one of claims 1-52, the recombinant virus of claim 53 or 54, or the pharmaceutical composition of claim 69, for the treatment of an individual in need thereof.
80. The method of claim 77 or use of claim 78 or 79, wherein the individual has a GPC3- positive disease.
81. The method or use of claim 80, wherein the GPC3-positive disease is cancer.
82. The method or use of claim 81, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, lung squamous cell carcinoma, ovarian carcinoma, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular nonseminomatous germ cell tumor, gastric carcinoma, and liposarcoma.
83. A method of reducing exhaustion of an engineered immune cell, comprising introducing to the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises the expression construct(s) of any one of claims 1-52.
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