Immune cell therapy

Abstract

The present disclosure provides compositions and methods for improving immune cell therapy. In a particular aspect, the present disclosure relates to one or more expression constructs expressing a chimeric antibody T cell receptor (TCR) construct (caTCR) and a chimeric stimulatory receptor (CSR) and a human c-Jun polypeptide, wherein both the caTCR and the CSR specifically target glypican 3 (GPC3).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 369,171, filed on July 22, 2022. The disclosure of the aforementioned provisional application is incorporated herein by reference in its entirety. Sequence Listing

[0002] This application contains a sequence listing, which has been submitted electronically in XML format. The sequence listing is incorporated herein 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] Over the past two decades, fundamental advances in immunology and tumor biology and 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 caused some significant anti-tumor responses (Blattman et al., Science. (2004) 305(5681):200-5). For example, the administration of ex vivo expanded naturally occurring tumor infiltrating lymphocytes (TILs) modulates objective response rates in the range of 50-70% in melanoma patients, including patients with large invasive tumors in multiple sites involving the liver, lungs, soft tissues, 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] The main limitation of the widespread application of TIL therapy is the difficulty in generating human T cells with anti-tumor potential. As an alternative, exogenous high-affinity TCRs can be introduced into normal autologous T cells of patients via T cell engineering. Adoptive transfer of these cells into patients with lymphocyte depletion has been shown to regulate tumor regression in cancers such as melanoma, colorectal cancer, 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 via MHC presentation; these antigens can be recognized by antigen-specific cytotoxic T cells even at lower density (Kunert, supra).

[0005] Several attempts have been made to engineer TCR molecules with antibody specificity and T cell receptor effector functions. In some of these methods, the variable domains and constant domains of TCR (e.g., αβTCR or γδTCR) are replaced by the variable domains and constant domains of antibodies against tumor antigens, thereby generating chimeric antibody TCRs, referred to as "abTCR" or "caTCR". See, for example, WO 2017 / 070608 and WO 2018 / 200582, the disclosures of which are incorporated herein by reference in their entirety. In one of the methods, a chimeric stimulatory receptor (CSR) is used in combination with caTCR to enhance the tumor killing efficacy of engineered T cells. Like a chimeric antigen receptor (CAR), CSR has an extracellular domain that binds to a target ligand (e.g., a tumor antigen) and an intracellular costimulatory domain, but unlike CAR, CSR does not have an intracellular primary immune cell signaling domain (which is typically an intracellular domain of a CD3ζ chain) or a functional primary immune cell signaling domain. CSR and caTCR can bind to different epitopes of the same target / antigen and act synergistically to enhance the cytotoxicity of engineered T cells. See, for example, WO 2018 / 200583, the disclosure of which is incorporated herein by reference in its entirety.

[0006] One challenge faced by T cell therapy is the lack of persistence of T cells in vivo due to a phenomenon called T cell exhaustion. See, for example, 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 significant changes in metabolic function, transcriptional programming, loss of effector function (e.g., reduced cytokine secretion and cytotoxicity), expression of multiple surface inhibitory receptors, and apoptosis. T cell exhaustion is attributed to constant antigen exposure, resulting in sustained TCR signaling, or tonic antigen-independent signaling via engineered antigen receptors on T cells (see, for example, Long, supra). In patients with cancer or chronic infection, for example, and in T cell therapy, prevention or reversal of T cell exhaustion has been sought as a means of enhancing T cell effectiveness. See, e.g., WO 2019 / 118902, the disclosure of which is incorporated herein by reference in its entirety.

[0007] Glypican 3 (GPC3, also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, GTR2-2) is a cell surface protein overexpressed in a variety of cancer types, including a variety of solid tumors, such as hepatocellular carcinoma (HCC), melanoma (Nakatsura T et al., Clin Cancer Res. (2004) 10(19):6612-21), squamous cell carcinoma of the lung (Yu X et al., Genet Mol Res. (2015) 14(3):10185-92), ovarian cancer (Stadlmann S et al., Int J 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 is still a medical need to develop treatments for various cancers, including cancers that overexpress GPC3.

[0008] Therefore, there remains a need for improved T cell therapies in which engineered T cells have higher and sustained tumor killing potency, especially improved T cell therapies in which T cells specifically target cancers that overexpress GPC3. Summary of the invention

[0009] The present disclosure provides compositions and methods for improving immune cell therapy. In some aspects, one or more expression constructs are provided herein, comprising one or more expression cassettes for expressing the following: 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulatory receptor (CSR), comprising: i) a ligand binding module that can bind or interact with GPC3; ii) a transmembrane module; and iii) a costimulatory immune cell signaling module that can provide 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.

[0010] In some embodiments, c-Jun is wild-type human c-Jun. In some embodiments, wild-type human c-Jun has at least about 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, c-Jun is a mutant human c-Jun. In some embodiments, the mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain. In some embodiments, c-Jun comprises: (i) S63A and S73A substitutions 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 compared to SEQ ID NO: 1.

[0011] In some embodiments, the GPC3 is cell surface-associated GPC3.

[0012] In some embodiments, the TCRM is derived from a human γ / δ TCR. In some embodiments, the caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have binding affinity to each other that stabilizes the caTCR. In some embodiments, the stabilizing module is selected from the group consisting of: a TCR constant domain, a C H 1-C L Module, C H 2-C H 2 modules, C H 3-C H 3 modules and C H 4-C H In some embodiments, the stabilizing module is derived from a human protein. H 1-C L The C included in the module L Derived from a kappa antibody light chain or a lambda antibody light chain. In some embodiments, the antigen binding moiety of the caTCR is Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv).

[0013] In some embodiments, the antigen binding moiety of the caTCR comprises: (i) a heavy chain variable domain (VHD) comprising a heavy chain complementary 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, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; H); and a light chain variable domain (VLD) comprising a light chain complementary 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, a LC-CDR2 comprising the amino acid sequence of GDN or a variant thereof comprising up to about 3 amino acid substitutions, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 3 amino acid substitutions. L ); (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to about 3 amino acid substitutions H and a V 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 L In some embodiments, the antigen binding moiety of the caTCR comprises: (i) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; Hand a V comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR2 comprising the amino acid sequence of GDN, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 L (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 H and a V 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 L or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 L .

[0014] In some embodiments, the antigen binding moiety of the caTCR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 24; H , and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:25 L (ii) a V comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 26 H , and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:27 L or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 28 H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:29 LIn some embodiments, the antigen binding moiety of the caTCR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L (ii) V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L .

[0015] In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a first TCRD and a second polypeptide chain comprising a second TCRD. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a V fused to a TCRD derived from a TCR delta chain and a second polypeptide chain. H , the second polypeptide chain comprises a V fused to a TCRD derived from a TCRγ chain L In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising a V fused to a TCRD derived from a TCRγ chain and a second polypeptide chain. H , the second polypeptide chain comprises a V fused to a TCRD derived from a TCRδ chain L. 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 having at least about 90% sequence identity with SEQ ID NO:30; and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least about 90% sequence identity with 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 having at least about 90% sequence identity with SEQ ID NO:10; and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least about 90% sequence identity with 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 having 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 having 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 having 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 having 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 having 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 having 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 having 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 having at least about 90% sequence identity to SEQ ID NO: 70.

[0016] In some embodiments, the transmembrane module of a CSR comprises a transmembrane domain derived from the transmembrane domain of CD30, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane module of a CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.

[0017] In some embodiments, the costimulatory immune cell signaling module is derived from the intracellular domain of the costimulatory receptor of TCR. In some embodiments, the costimulatory receptor is selected from the group consisting of CD30, CD28, 4-IBB, OX40, ICOS, CD27 and CD40. In some embodiments, the costimulatory immune cell signaling module of CSR is derived from human CD30. In some embodiments, the costimulatory immune cell signaling module of human CD30 includes the amino acid sequence of SEQ ID NO:43. In some embodiments, the costimulatory immune cell signaling module of human CD30 includes the amino acid sequence of SEQ ID NO:44. In some embodiments, the costimulatory immune cell signaling module and the transmembrane domain of CSR are derived from CD30 (e.g., human CD30).

[0018] In some embodiments, the ligand binding moiety of a CSR comprises: (i) a heavy chain variable domain (VHD) comprising a heavy chain complementary 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, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; H ); and a light chain variable domain (VLD) comprising a light chain complementary 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, a LC-CDR2 comprising the amino acid sequence of GDN or a variant thereof comprising up to about 3 amino acid substitutions, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 3 amino acid substitutions. L); (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to about 3 amino acid substitutions H and a V 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 L In some embodiments, the ligand binding module of the CSR comprises: (i) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a LC-CDR2 comprising the amino acid sequence of GDN, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 L (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 Hand a V 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 L or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 L .

[0019] In some embodiments, the ligand binding module of the CSR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 24; H , and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:25 L (ii) a V comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 26 H , and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:27 L or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 28 H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:29 L In some embodiments, the ligand binding module of the CSR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24; H and V comprising the amino acid sequence of SEQ ID NO:25 L (ii) V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 Hand V comprising the amino acid sequence of SEQ ID NO: 29 L .

[0020] In some embodiments, the ligand binding module CSR comprises: (i) SEQ ID NO:32 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:32; (ii) SEQ ID NO:33 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:33; or (iii) SEQ ID NO:34 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:34.

[0021] In some embodiments, (i) the antigen binding moiety of the caTCR comprises: (a) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20. H and (b) a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. L and (ii) the ligand binding module of the CSR comprises: (a) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; H and (b) a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LC-CDR2 comprising the amino acid sequence of YDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17 L .

[0022] In some embodiments, the antigen binding moiety of the caTCR comprises a Fab that binds GPC3, wherein the ligand binding moiety of the CSR comprises a scFv that binds GPC3, and wherein the transmembrane moiety of the CSR and the co-stimulatory immune cell signaling moiety 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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29L ; and (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L 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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; and (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L 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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; and (ii) the scFv comprises a V comprising an amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; and (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; and (ii) the scFv comprises a V comprising an amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 LIn 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; and (ii) the scFv comprises a V comprising an amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L ; and (ii) the scFv comprises a V comprising an amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L ; and (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L ; and (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L .

[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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29L ; 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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L; 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L ; 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 V comprising the amino acid sequence of SEQ ID NO: 24. H and V comprising the amino acid sequence of SEQ ID NO:25 L ; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO:34.

[0025] In some embodiments, the construct is a viral vector. In some embodiments, the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex virus vector, and an Epstein-Barr viral vector.

[0026] In some embodiments, the expression construct comprises a polycistronic expression cassette for expressing caTCR, CSR, and c-Jun.

[0027] In some aspects, provided herein is a multicistronic 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 a human γ / δ TCR; b) a chimeric stimulatory receptor (CSR) comprising: i) a ligand binding module that can bind or interact with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from the intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 28 and 29, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 28 and 29, and the coding sequence of SEQ ID NO: 32. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 28 and 29, and the coding sequence of SEQ ID NO: 34. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 26 and 27, and the coding sequence of SEQ ID NO: 32. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 26 and 27, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 26 and 27, and the coding sequence of SEQ ID NO: 34. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 24 and 25, and the coding sequence of SEQ ID NO: 32. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 24 and 25, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequences of SEQ ID NOs: 24 and 25, and the coding sequence of SEQ ID NO: 34.

[0028] In some embodiments, the coding sequence is separated in frame by a 2A coding sequence or by an internal ribosome entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF-1α promoter. In some embodiments, the construct is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex virus vector, and an Epstein-Barr virus vector. In some embodiments, a recombinant virus is provided herein, comprising a polycistronic expression construct as in any of the foregoing embodiments. In some embodiments, the polycistronic expression construct is a lentiviral vector.

[0029] In some aspects, a method for engineering immune cells is provided herein, comprising: (a) providing a starting cell population; (b) introducing an expression construct as in any of the foregoing technical solutions or a recombinant virus as in any of the foregoing technical solutions into the starting cell population; (c) optionally selecting cells expressing caTCR, CSR and c-Jun; and (d) obtaining engineered immune cells from the cells of step (b) or (c). In some embodiments, the immune cells are T cells. In some embodiments, T cells are selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells and suppressor T cells. In some embodiments, the 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 multipotent or pluripotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells.

[0030] In some aspects, a human cell population is provided herein, comprising an expression construct of any of the foregoing embodiments or a recombinant virus of any of the foregoing embodiments. In some embodiments, the human cells are immune cells. In some embodiments, the immune cell population is obtained by a method as in any of the foregoing 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 less exhaustion markers than corresponding cells that do not overexpress c-Jun. In some embodiments, the exhaustion markers are 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 an expression construct as in any of the preceding embodiments, a recombinant virus as in any of the preceding embodiments, or a cell as in any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0032] In some aspects, provided herein is a method of killing a target cell, comprising contacting the target cell with a pharmaceutical composition as in any of the foregoing embodiments under conditions that allow killing of the target cell by an immune cell, wherein the target cell expresses GPC3. In some embodiments, the immune cell expresses a lower amount of an exhaustion marker when contacted with the target cell than a corresponding immune cell that does not contain an exogenous nucleic acid molecule that causes overexpression of c-Jun. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8 + T cells. In some embodiments, the target cell is a cancer cell. In some embodiments, the costimulatory immune cell signaling module in the CSR is derived from human CD30, and the immune cell expresses a lesser amount of exhaustion markers than the corresponding immune cells of the CSR engineered to express the costimulatory immune cell signaling module 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, a method for treating an individual in need is provided herein, comprising administering to the individual a pharmaceutical composition as in any of the foregoing embodiments. In some aspects, the use of an expression construct of any of the foregoing embodiments, a recombinant virus of any of the foregoing embodiments, or a pharmaceutical composition of any of the foregoing embodiments is provided herein for the manufacture of a drug for treating an individual in need. In some aspects, the use of an expression construct of any of the foregoing embodiments, a recombinant virus of any of the foregoing embodiments, or a pharmaceutical composition of any of the foregoing embodiments is provided herein for the treatment of an individual in need. In some embodiments, the individual suffers from 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, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, and liposarcoma.

[0034] In some aspects, provided herein is a method of reducing exhaustion of an engineered immune cell, comprising introducing into the engineered immune cell an exogenous nucleic acid molecule that increases expression of c-Jun in the cell, wherein the engineered immune cell comprises an expression construct as in any one of the preceding embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings illustrate certain embodiments having features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0036] Figure 1A Schematic representation of an exemplary caTCR and CSR pair (top) and two expression constructs used for in vivo efficacy studies of T cells described herein (bottom). Construct 1 encodes an anti-GPC3 caTCR ("anti-GPC3-caTCR") plus an anti-GPC3 CSR containing a CD30-derived co-stimulatory domain ("anti-GPC3-CD30-CSR"), while construct 2 encodes "anti-GPC3-caTCR" + "anti-GPC3-CD30-CSR" and "c-Jun" polypeptide.

[0037] Figure 1B Schematic representation of the design for in vivo efficacy studies of T cells expressing anti-GPC3-caTCR and anti-GPC3-CD30-CSR in the presence or absence of c-Jun overexpression in two different mouse models.

[0038] Figure 2 Figure 1 shows the in vivo tumor killing effect of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells in the HepG2-luc mouse model in the presence or absence of c-Jun overexpression. T cells were isolated from two healthy human donors. For each T cell type, two doses (5×10 6 and 2×10 6 ).

[0039] Figure 3 Graph showing the in vivo tumor killing effect of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells in the presence or absence of c-Jun overexpression in the HepG2-luc mouse model in tumor re-challenge studies, in which animals were re-implanted with tumor cells after initial tumor regression.

[0040] FIG. 4A to FIG. 4C Graph showing peripheral blood T cell counts in HepG2-luc and Hep3B mice injected with anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells in the presence or absence of c-Jun overexpression. Figure 4A In , T cells were isolated from donor A and the mouse model was HepG2-luc; Figure 4B In , T cells were isolated from donor B and the mouse model was HepG2-luc; Figure 4C In this study, T cells were isolated from donor C and the mouse model was Hep3B.

[0041] Figure 5 Graph showing the in vivo tumor killing effect of anti-GPC3-caTCR plus anti-GPC3-CD30-CSR T cells in the presence or absence of c-Jun overexpression in the Hep3B mouse model. DETAILED DESCRIPTION OF THE INVENTION

[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 stimulatory receptor (CSR; also referred to herein as a "chimeric signaling receptor"), and a c-Jun polypeptide (e.g., a human c-Jun polypeptide). Both the caTCR and the CSR specifically bind to or interact with GPC3 (also referred to herein as "anti-GPC3 caTCR" and "anti-GPC3 CSR," respectively). The anti-GPC3 caTCR comprises an antigen binding module that specifically binds to GPC3, and a T cell receptor module (TCRM) that is 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 that is capable of providing a stimulatory signal to an immune cell and does not comprise a functional primary T cell signaling sequence. The GPC3 targeted by the anti-GPC3 caTCR and anti-GPC3 CSR 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 cancer, such as a cancer of the gastrointestinal system, for example, gastric cancer or liver cancer. The anti-GPC3 caTCR regulates through a naturally occurring mechanism that controls TCR activation, while the anti-GPC3 CSR enhances the immune response mediated by the anti-GPC3 caTCR. c-Jun expression helps maintain the active state of T cells by, for example, alleviating or preventing T cell exhaustion.

[0043] The engineered immune cells of the present invention such as T cells show sustained and effective cytotoxicity for tumor cells carrying targets. Compared to T cells that do not overexpress c-Jun (e.g., nucleic acid sequences introduced via exogenous encoding c-Jun), the engineered T cells of the present invention show less signs of T cell exhaustion. The engineered cells may have one or more of the following features: (i) they do not have increased expression of exhaustion markers PD-1, TIM-3 and / or LAG-3 over time; (ii) they have a reduced apoptosis rate; (iii) they increase the formation of memory cells and / or maintain memory markers (e.g., CCR7 and CD45RA); (iv) they have enhanced cytotoxicity; (v) they show improved recognition of tumor targets with low surface antigens; (vi) they have enhanced proliferation in response to antigens; (vii) they maintain survival and function after repeated antigen stimulation; and (viii) they show increased tumor infiltration ability.

[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 inventors unexpectedly discovered that c-Jun overexpression significantly reduced exhaustion of T cells engineered to express anti-GPC3 caTCR and CD30-based anti-GPC3 CSR. I. Definitions

[0045] The term "antibody" or "antibody portion" includes full-length antibodies and antigen-binding fragments thereof. Full-length antibodies contain two heavy chains and two light chains. The variable regions (i.e., variable domains) of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are inserted between the side segments called framework regions (FRs), which are more conservative than CDRs and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0046] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), an scFv dimer (a bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody of a camel, 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 as that bound by a parent antibody or a parent antibody fragment (e.g., a parent scFv). In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted to a framework region from one or more different human antibodies.

[0047] As used herein, the term "specifically binds" or "is specific for" refers to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, which determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody portion that specifically binds to a target (which may be an epitope) is an antibody portion that binds to the target with greater affinity, avidity, ease, and / or duration than it binds to other targets. In some embodiments, the antibody portion that specifically binds to an antigen has a binding affinity that is at least about 10 times greater than its binding affinity for other targets in its reaction to one or more antigenic determinants of the antigen (e.g., a cell surface antigen or a peptide / MHC protein complex).

[0048] The term "T cell receptor" or "TCR" refers to a heterodimeric receptor composed of paired αβ or γδ chains on the surface of T cells. Each α, β, γ and δ chain consists of two Ig-like domains: a variable domain (V) that confers antigen recognition via a complementary determining region (CDR), followed by a constant domain (C) anchored to the cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region binds to the constant subunit of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between antigenic peptides that bind to proteins encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012), xix, p. 868).

[0049] The term "TCR-associated signaling molecule" refers to a molecule with a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) that is part of the TCR-CD3 complex. TCR-associated signaling molecules include CD3γε, CD3δε, and ζζ (also referred to as CD3ζ or CD3ζζ).

[0050] "Activation" as used herein with respect to cells expressing CD3 refers to a state of a cell that has been sufficiently stimulated to induce a detectable increase in downstream effector functions of the CD3 signaling pathway, including but not limited to cell proliferation and cytokine production.

[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 that make up the protein. For example, a transmembrane module of a dimeric receptor may refer to the transmembrane portion of each polypeptide chain of the receptor. A module may also refer to the related portion of a single polypeptide chain. For example, a transmembrane module of a monomeric receptor may refer to the transmembrane portion of a single polypeptide chain of the receptor. A module may also include only a single portion of a polypeptide.

[0052] As used herein, the term "isolated nucleic acid" means a nucleic acid of genomic, cDNA, or synthetic origin, or some combination thereof, which, by virtue of its origin, (1) is not associated with all or a portion of a polynucleotide with which the "isolated nucleic acid" occurs in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur as part of a larger sequence in nature.

[0053] As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen combining sites found within the variable regions of the heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. 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: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc 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: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309: 657-670 (2001) describe, wherein the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to an antibody or a CDR or variant thereof of a transplanted antibody is intended to fall within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined by each of the above cited references are set forth 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 entirety for use in the present invention and may be included 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. 1 The residue numbering follows the nomenclature of Kabat et al. 2 The residue numbering follows the nomenclature of Chothia et al. 3 The residue numbering follows the nomenclature of MacCallum et al. 4 The residue numbering follows the nomenclature of Lefranc et al. 5 The residue numbering follows the nomenclature of Honegger and Plückthun mentioned above.

[0054] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the biological activity of the present invention (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0055] The term "semisynthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has one or more naturally occurring sequences and one or more non-naturally occurring (ie, synthetic) sequences.

[0056] The term "fully synthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has a fixed, naturally occurring V H / V L The framework pairs are non-naturally occurring (ie, synthetic) sequences rather than all six CDRs of both the heavy and light chains.

[0057] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region and a light chain variable region in tight, non-covalent association. The two domains fold to obtain six hypervariable loops (3 loops each for the heavy chain and light chain), which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even if a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can recognize and bind antigen, the affinity is lower than that of the entire binding site.

[0058] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") is a fragment comprising a V domain linked in a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H With V LA polypeptide linker between domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0059] The "humanized" form of non-human (e.g., rodent) antibodies is a chimeric antibody containing minimal sequences derived from non-human antibodies. To a large extent, humanized antibodies are human immunoglobulins (recipient antibodies), wherein the hypervariable region (HVR) residues from the recipient are replaced with residues from the hypervariable region of a non-human species (donor antibody) (such as mice, rats, rabbits, or non-human primates with desired antibody specificity, affinity, and ability). In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will include substantially all variable domains in at least one and usually two variable domains, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins and all or substantially all FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional 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 the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecules are "homologous" at that position. The "homology percentage" or "sequence identity percentage" between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions multiplied by 100, and any conservative substitution is considered as part of the sequence identity. For example, if there are 6 matches or homologs in 10 positions in the two sequences, the two sequences are 60% homologous. For example, the DNA sequence ATTGCC shares 50% homology with TATGGC. In general, comparisons are made when aligning the two sequences to obtain maximum homology. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the skill range of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring comparison, including any algorithm required for achieving maximum comparison over the full length of the compared sequences. However, for purposes herein, the sequence comparison computer program MUSCLE is used to generate amino acid sequence identity % values ​​(Edgar, RC, Nucleic Acids Research 32 (5): 1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5 (1): 113, 2004).

[0061] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Insofar as nucleotide sequences encoding proteins may contain introns in some forms, the phrase "nucleotide sequence encoding the protein or RNA" may also include introns.

[0062] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, thereby causing the latter to be expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. In general, operably linked DNA sequences are contiguous and, if necessary, are linked so that two protein coding regions are 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 can activate transcription of an operably linked nucleic acid under a specific set of conditions, such as in the presence of an inducing agent or conditions that activate the promoter and / or relieve the repression of the promoter.

[0064] As used herein, the term "cell engineering" or "cell modification" (including derivatives thereof) refers to the targeted modification of cells, such as the immune cells disclosed herein.

[0065] As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: relieving one or more symptoms caused by the disease, reducing the scope of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread (e.g., metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, causing disease remission (partial or complete), reducing the dose of one or more other agents required for treating the disease, delaying disease progression, improving or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses reducing the pathological results of the disease (such as tumor volume in cancer). The methods of the present invention encompass any one or more of these therapeutic aspects.

[0066] The terms "recurrence," "relapse," or "relapsed" refer to the return of cancer or disease after clinical assessment of disease disappearance. The diagnosis of distant cancer metastasis or local recurrence may be considered a recurrence.

[0067] The term "refractory" or "resistant" refers to a cancer or disease that does not respond to treatment.

[0068] An "effective amount" of a caTCR or a composition comprising a caTCR as disclosed herein is an amount sufficient to carry out a specific stated purpose. An "effective amount" can be determined empirically and by known methods relevant to the stated purpose.

[0069] The term "therapeutically effective amount" refers to the amount of a caTCR or a composition comprising a caTCR as disclosed herein that is effective to "treat" an individual's disease or condition. In the case of cancer, a therapeutically effective amount of a caTCR or a composition comprising a caTCR as disclosed herein can reduce the number of cancer cells; reduce tumor size or weight; inhibit (i.e., slow down to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. Insofar as a caTCR or a composition comprising a caTCR as disclosed herein can prevent growth and / or kill existing cancer cells, it can be cell growth inhibitory 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 increases the patient's progression-free survival. In the case of infectious diseases such as viral infections, a therapeutically effective amount of a caTCR or a composition comprising a caTCR as disclosed herein can reduce the number of cells infected with pathogens; reduce the production or release of antigens derived from pathogens; inhibit (i.e., slow down to some extent and preferably prevent) the spread of pathogens to uninfected cells; and / or alleviate to some extent one or more symptoms associated with infection. In some embodiments, a therapeutically effective amount is an amount that prolongs patient survival.

[0070] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that has no adverse effects biologically or otherwise, i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any significant adverse biological effects or interacting in a harmful manner with any other component in the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the necessary standards of toxicology and manufacturing testing and / or are included in the Inactive Ingredient Guide established by the US Food and Drug Administration.

[0071] It is to be understood that the embodiments of the present invention described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0072] References herein to "about" a value or parameter include (and describe) variations to that value or parameter itself. For example, descriptions referring to "about X" include descriptions of "X."

[0073] As used herein, reference to "not a value or parameter" generally means and describes "except for a value or parameter." For example, a method is not used to treat type X cancer means that the method is used to treat types of cancer other than type 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 conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. Although methods and materials similar to or equivalent to those described herein can be used to practice or test the present disclosure, exemplary methods and materials are described below. In the event of a conflict, the present invention (including definitions) shall prevail. In general, the nomenclature and techniques used in conjunction with immunology, medicine, pharmaceutical and medicinal chemistry, and cell biology described herein are well known and commonly used in the art. In addition, unless the context otherwise requires, singular terms should include plural terms and plural terms should include singular terms. Throughout this specification and embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" should be understood to imply the inclusion of stated integers or integer groups, but do not exclude any other integers or integer groups. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety. Although multiple documents are cited herein, this citation does not constitute a license for any of these documents to form part of the common general knowledge in the art. As used herein, the term "substantially" or "approximately" when applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term refers to a range of values ​​within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1% in either direction (greater than or less than) of the stated reference value. 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 antigen binding moiety that specifically binds to GPC3), a CSR (e.g., a CSR comprising a ligand binding moiety that can bind to or interact 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 (the (one or more) expression constructs are also referred to herein as "(one or more) caTCR+CSR+c-Jun expression constructs"). 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 the terms "express / expressing" and "encode / encoding" are used interchangeably herein when used to describe an expression construct or an expression cassette.

[0077] In some aspects, provided herein are one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding moiety 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding moiety that can bind or interact with GPC3; ii) a transmembrane moiety; and iii) a costimulatory immune cell signaling moiety that can provide a costimulatory signal to the immune cell, wherein the ligand binding moiety and the costimulatory immune cell signaling moiety 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 construct is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex virus vector, and an Epstein-Barr virus vector. In some embodiments, the expression construct expresses a polycistronic expression cassette for expressing anti-GPC3 caTCR, anti-GPC3 CSR, and c-Jun. In some embodiments, the TCRM of the anti-GPC3 caTCR is derived from human γ / δTCR. In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises a transmembrane domain 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 wild-type human c-Jun polypeptide.

[0078] 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 moiety comprising: (1) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; Hand (2) a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. L ; 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding module comprising: (1) a V comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 H and (2) a V 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. L ; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to the immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3 caTCR comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H and V comprising the amino acid sequence of SEQ ID NO: 29 L . 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 stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the ligand binding module of the anti-GPC3 CSR comprises a V comprising the amino acid sequence of SEQ ID NO:26. Hand V comprising the amino acid sequence of SEQ ID NO: 27 L 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 a transmembrane domain 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 wild-type 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 are one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding moiety 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding moiety comprising a scFv that can bind or interact with GPC3; ii) a transmembrane moiety derived from CD30; and iii) a costimulatory immune cell signaling moiety derived from CD30 that can provide a costimulatory signal to an 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 (VL) comprising the amino acid sequence of SEQ ID NO: 28. H ) and a light chain variable domain (V L In some embodiments, the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L 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 multicistronic 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 a human γ / δ 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 the intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 28 and 29, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 30 and 31, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 10 and 5, and the coding sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 28 and 29, and the coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 30 and 31, and the coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 10 and 5, and the coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 30 and 31, and the coding sequence of SEQ ID NO: 16. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NOs: 10 and 5, and the coding sequence of 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 of SEQ ID NO:2. In some embodiments, the coding sequence is separated in frame by a 2A coding sequence or by an internal ribosome entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF-1α 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 encode chimeric antibody T cell receptor (caTCR) constructs (also referred to herein as "anti-GPC3 caTCRs") comprising an antigen binding moiety that specifically binds to GPC3. Exemplary caTCRs are provided in PCT / US2016 / 058305, the contents of which are incorporated herein by reference in their entirety. Anti-GPC3 caTCRs can specifically bind to GPC3 and can recruit at least one TCR-associated signaling molecule (such as CD3 δε, CD3 γε, and / or ζζ). In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety that specifically binds to GPC3 bound to the surface of a cell. In some embodiments, an anti-GPC3 caTCR is provided, comprising a) an antigen binding moiety that specifically binds to GPC3; and b) a TCR module (TCRM) comprising a first TCR domain (TCRD) and a second TCRD, the first TCRD comprising a first TCR transmembrane domain (TCR-TM) derived from one of the transmembrane domains of a naturally occurring TCR (such as an αβTCR or a γδTCR), the second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring TCR (such as an αβTCR or a γδTCR), wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule (such as CD3δε, CD3γε and / or ζζ), and wherein the antigen binding moiety is linked to the first and / or second TCRD. In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing moiety comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain 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 of the features described in this section. It is intended that any of the features of the various components of the anti-GPC3 caTCRs described herein (e.g., antigen binding moiety, TCRD, TCR-TM, spacer module, stabilization module, T cell co-stimulatory sequence, various linkers, etc.) may be combined with each other, with any of the features of the anti-GPC3 CSRs, and with c-Jun, as if each combination were individually described.

[0083] In some embodiments, the antigen binding moiety (such as an antibody portion) can specifically bind 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 greater) times greater than its binding affinity for other molecules; or b) a binding affinity that is at least about 10, 20, 30, 40, 50, 750, 1000, or greater.d Does not exceed its K for binding to other molecules d The binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). d It can be determined by methods known in the art, such as surface plasmon resonance (SPR) using, for example, a Biacore instrument, or kinetic exclusion analysis (KinExA) using, for example, a Sapidyne instrument.

[0084] In some embodiments, the stabilizing module comprises a first stabilizing domain and a second stabilizing domain. In some embodiments, the first stabilizing domain and the second stabilizing domain have binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilizing module is located between the antigen binding module and the TCRM. In some embodiments, the stabilizing module is derived from the antibody portion. In some embodiments, the stabilizing module is derived from the antibody constant domain. In some embodiments, the stabilizing module is derived from the antibody heavy chain constant domain. In some embodiments, the antibody heavy chain constant domain contained in the stabilizing module is derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain, optionally human. In some embodiments, the antibody heavy chain constant domain contained in the stabilizing 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 antibody light chain constant domain contained in the stabilizing module is derived from a κ or λ light chain, optionally human. In some embodiments, the stabilizing module is derived from an antibody light chain constant domain. In some embodiments, the antibody light chain constant domain contained in the stabilizing 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 stabilizing domains comprise one or more modifications that do not substantially change their binding affinity to each other. In some embodiments, the first and / or second stabilizing domains comprise one or more modifications that increase their binding affinity to each other and / or introduce non-naturally occurring disulfide bonds. In some embodiments, the stabilizing module comprises a knob-into-hole modification (see, e.g., Carter P. J Immunol Methods. 248: 7-15, 2001). For example, in some embodiments, the stabilizing module comprises an antibody constant domain region (e.g., C H 3 domains). In some embodiments, the stabilizing module comprises an antibody constant domain region modified by electrostatic steering to enhance its binding (see, e.g., WO2006106905 and Gunasekaran K et al. J Biol Chem. 285: 19637-46, 2010). In some embodiments, the first stabilizing domain and the second stabilizing domain are connected by a disulfide bond.

[0085] Examples of stabilizing domains include the Fc region; the hinge region; the C H 2 domains; C H 3 domains; C H 4 domains; C H 1 domain or C Ldomain; TCR constant domain; leucine zipper domain (e.g., jun / fos leucine zipper domain, see, e.g., Kostelney et al., J. Immunol, 148:1547-1553, 1992; or yeast GCN4 leucine zipper domain); isoleucine zipper domain; dimerization region of a dimerizing cell surface receptor (e.g., interleukin-8 receptor (IL-8R); or integrin heterodimers, such as LFA-1 or GPIIIb / IIIa); secreted dimerizing ligand dimerization region of the protein (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); coiled-coil dimerization domain (coiled coildimerization domain) (see, e.g., 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., about one, two or three to about ten cysteine ​​residues), such that (one or more) disulfide bonds can be formed between the polypeptide and a second polypeptide comprising at least one cysteine ​​residue.

[0086] In some embodiments, the TCRM described herein includes a first T cell receptor domain (TCRD) and a second TCRD, the first TCRD includes a first TCR transmembrane domain (TCR-TM), the second TCRD includes a second TCR-TM, wherein TCRM promotes the recruitment of at least one TCR-related signal transduction molecule. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, both TCR-TMs are non-naturally occurring. In some embodiments, the first TCR-TM is derived from one of the transmembrane domains of a T cell receptor (such as αβTCR or γδTCR), and the second TCR-TM is derived from another transmembrane domain of a T cell receptor. In some embodiments, the TCRM is compared to a TCRM comprising a T cell receptor transmembrane domain, and enhances the recruitment of at least one TCR-related signal transduction molecule. The recruitment of TCR-related signal transduction molecules can be determined by methods known in the art, such as FACS analysis or CD3 subunits co-immunoprecipitated with caTCR for TCR-CD3 complex surface expression. For example, in some embodiments, the first TCR-TM of the TCRM described herein comprises a transmembrane domain of a TCR alpha chain (e.g., GenBank Accession No.: CCI73895) or a variant thereof, is substantially composed of, or is composed of, and the second TCR-TM of the TCRM comprises a transmembrane domain of a TCR beta chain (e.g., GenBank Accession No.: CCI73893) or a variant thereof, is substantially composed of, or is composed of. In some embodiments, the first TCR-TM comprises a transmembrane domain of a TCR delta chain (e.g., GenBank Accession No.: AAQ57272) or a variant thereof, is substantially composed of, or is composed of, and the second TCR-TM comprises a transmembrane domain of a TCR gamma chain (e.g., GenBank Accession No.: AGE91788) or a variant thereof, is substantially composed of, or is composed of. In some embodiments, the first and second TCR-TMs of the TCRRMs described herein comprise, consist essentially of, or consist of the following: a transmembrane domain of a TCR α chain constant domain (e.g., SEQ ID NO: 35) or a variant thereof and a transmembrane domain of a TCR β 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 following: a transmembrane domain of a TCR δ chain constant domain (e.g., SEQ ID NO: 37) or a variant thereof and a transmembrane domain of a TCR γ 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 NO: 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, respectively, or variants thereof. Variants of transmembrane domains include, but are not limited to, transmembrane domains having one or more amino acid substitutions compared to a reference sequence. In some embodiments, the variant transmembrane domain comprises no more than 5 amino acid substitutions compared to a reference sequence.

[0087] In some embodiments, the first TCRD further comprises a first connecting peptide at the amino terminus of the transmembrane domain and / or the second TCRD further comprises a second connecting peptide at the amino terminus of the transmembrane domain. In some embodiments, the first connecting peptide comprises all or part of a connecting peptide of a TCR subunit from which the first TCR-TM is derived, or a variant thereof, and / or the second connecting peptide comprises all or part of a connecting peptide of a 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 the following, respectively: all or part of a connecting peptide of a TCR α chain constant domain, or a variant thereof, and all or part of a connecting peptide of a TCR β chain constant domain, or a variant thereof. In some embodiments, the first and / or second connecting peptides comprise, consist essentially of, or consist of the following, respectively: all or part of a connecting peptide of a TCR δ chain constant domain, or a variant thereof, and all or part of a connecting peptide of a TCR γ chain constant domain, or a variant thereof. In some embodiments, the first TCRD is further included in the first TCR intracellular domain at the carboxyl end of the first TCR-TM and / or the second TCRD is further included in the second TCR intracellular domain at the carboxyl end of the second TCR-TM. In some embodiments, the first TCR intracellular domain includes all or part of the intracellular domain of the TCR subunit derived from the first TCR-TM or its variant, and / or the second TCR intracellular domain includes all or part of the intracellular domain of the TCR subunit derived from the second TCR-TM or its variant. In some embodiments, the first TCRD is a fragment of a chain of a naturally occurring TCR or its variant, and / or the second TCRD is a fragment of another chain of a naturally occurring TCR or its variant. In some embodiments, at least one of the TCRDs is non-naturally occurring. The non-naturally occurring TCR domain can be the corresponding domain of a naturally occurring TCR, which is modified by substitution of one or more amino acids and / or by replacing a portion of the corresponding domain with a portion of a similar domain from another TCR.

[0088] In some embodiments, the first TCR-TM comprises the following, consists essentially of the following, or consists of the following: SEQ ID NO: 3 or SEQ ID NO: 3 comprises at least about at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity. In some embodiments, the second TCR-TM comprises the following, consists essentially of the following, or consists of the following: SEQ ID NO: 4 or SEQ ID NO: 4 comprises at least about at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity. In some embodiments, the first and second TCRDs are connected by disulfide bonds. In some embodiments, the first and second TCRDs are connected by disulfide bonds between residues in the first connecting peptide and residues in the second connecting peptide. In some embodiments, TCRM is capable of recruiting at least one TCR-associated signaling molecule selected from the group consisting of CD3δε, CD3γε and ζζ. In some embodiments, TCRM is capable of recruiting each of CD3δε, CD3γε and ζζ to form a caTCR-CD3 complex (i.e., promoting caTCR-CD3 complex formation).

[0089] In some embodiments, the antigen binding moiety is an antibody portion selected from the group consisting of: a full-length antibody, Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv). In some embodiments, the antigen binding moiety is a Fab or Fab' antibody portion. In some embodiments, the antigen binding moiety is a scFv antibody portion. In some embodiments, the antibody portion is a fully human antibody portion, a semisynthetic antibody portion having a human antibody framework region, or a humanized antibody portion.

[0090] In some embodiments, the antigen binding moiety comprises an anti-GPC3 V H The first antigen binding domain of the antibody domain and the anti-GPC3 V L In some embodiments, the anti-GPC3 V H Antibody domains and anti-GPC3 V L The antibody domains and CDRs are derived from the same antibody portion. H Antibody domains and anti-GPC3 V L Some of the antibody domain CDRs are derived from different antibody portions. H Antibody domains and / or anti-GPC3 V LThe antibody domain is a human antibody domain, a humanized antibody domain, a chimeric antibody domain, a semisynthetic antibody domain, or a fully synthetic antibody domain.

[0091] In some embodiments, the antigen binding moiety is an antibody portion comprising a specific CDR sequence derived from one or more antibody portions (such as a monoclonal antibody) that are specific for GPC3 or certain variants of such sequences that comprise one or more amino acid substitutions. In some embodiments, the amino acid substitutions in the variant sequence do not substantially reduce the ability of the antigen binding moiety to bind to GPC3. Changes that substantially improve the binding affinity of the target antigen or affect some other property (such as specificity for and / or cross-reactivity with related variants of GPC3) are also contemplated.

[0092] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety as described herein connected to a TCRM described herein, optionally including a stabilizing moiety. For example, in some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety connected to the N-terminus of one or both of the TCRDs. In some embodiments, the anti-GPC3 caTCR comprises a stabilizing moiety between the TCRM and the antigen binding moiety. 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 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70, including any range between these values) amino acids in length. In some embodiments, the anti-GPC3 caTCR further comprises one or more auxiliary intracellular domains. In some embodiments, one or more auxiliary intracellular domains are located at the carboxyl end of the first TCRD and / or the second TCRD. In some embodiments, one or more auxiliary intracellular domains are located 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, one or more auxiliary intracellular domains individually include TCR costimulatory domains. In some embodiments, TCR costimulatory domains include all or part of the intracellular domain of immune costimulatory molecules (such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically bound to 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 a first and second TCR-TM of a transmembrane domain derived from a TCR (such as an αβTCR or a γδTCR), wherein the TCRM is capable of recruiting at least one TCR-related signaling molecule. In some embodiments, the antigen binding module is connected 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 connected to the amino terminus of one or two 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 at the amino terminus of the TCR-TM. In some embodiments, the TCRM further comprises at least one TCR intracellular domain, and the at least one TCR intracellular domain comprises a sequence from a TCR intracellular domain at the carboxyl terminus of the TCR-TM. In some embodiments, TCRM comprises a TCRD of a fragment derived from a TCR chain. In some embodiments, at least one of the TCRDs is non-naturally present. In some embodiments, caTCR further comprises at least one auxiliary intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30 or CD40) at the carboxyl end of TCR-TM. In some embodiments, caTCR lacks a co-stimulatory signaling sequence. In some embodiments, the antigen binding moiety is an antibody portion that specifically binds to GPC3 ("anti-GPC3 antibody portion"). In some embodiments, the anti-GPC3 antibody portion comprises anti-GPC3 V H Antibody domains and anti-GPC3 V L In some embodiments, the anti-GPC3 antibody portion is a human antibody domain, a humanized antibody domain, a chimeric antibody domain, a semisynthetic antibody domain, or a fully synthetic antibody domain. In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilizing module comprises at least one disulfide bond connecting the stabilizing domains. In some embodiments, the first stabilizing domain and the second stabilizing domain comprise antibody domains (such as C H 1 and C LAntibody domain) or its variant. In some embodiments, TCRM is capable of recruiting at least one TCR-related signaling molecule selected from the group consisting of CD3δε, CD3γε and ζζ. In some embodiments, TCRM allows the recruitment of at least one TCR-related signaling molecule to be enhanced compared to TCRM comprising a T cell receptor transmembrane domain. In some embodiments, TCRM promotes the formation of caTCR-CD3 complexes. In some embodiments, there is a spacing module between any two anti-GPC3 caTCR modules or domains. In some embodiments, anti-GPC3caTCR is a heteropolymer, such as a heterodimer. For example, in some embodiments, anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a first TCRD and a second polypeptide chain comprising a second TCRD, wherein the antigen binding module is connected to the first and / or second polypeptide chain.

[0094] In some embodiments, the anti-GPC3 caTCR described herein specifically binds to GPC3 and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the TCR transmembrane domains; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of the TCR, wherein the first and second TCRDs form a TCRM capable of recruiting at least one TCR-associated signaling molecule; and b) an antigen binding module that specifically binds to GPC3, wherein the antigen binding module is connected to the first and / or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the TCR is an αβTCR and the first and second TCR-TMs are derived from the TCRα and β subunit transmembrane domains. In some embodiments, the TCR is a γδTCR, and the first and second TCR-TMs are derived from the TCRγ and δ 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 includes all or part of the connecting peptide of the TCR subunit derived from the first TCR-TM or its variant, and / or the second connecting peptide includes all or part of the connecting peptide of the TCR subunit derived from the second TCR-TM or its variant. In some embodiments, the first and second connecting peptides are connected by a disulfide bond. In some embodiments, the first TCRD further includes the first TCR intracellular domain, and / or the second TCRD further includes the second TCR intracellular domain. In some embodiments, the first TCR intracellular domain includes the sequence of the intracellular domain of the TCR subunit derived from the first TCR-TM, and / or the second TCR intracellular domain includes the sequence of the intracellular domain of the TCR subunit derived from the second TCR-TM. In some embodiments, the first TCRD is a fragment of the TCR subunit derived from the first TCR-TM, and / or the second TCRD is a fragment of the TCR subunit derived from the second TCR-TM. In some embodiments, the anti-GPC3 caTCR further comprises at least one auxiliary intracellular domain comprising a T cell co-stimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40). In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first stabilizing domain and the second stabilizing domain are connected by a disulfide bond. In some embodiments, the first stabilizing domain and the second stabilizing domain comprise an antibody domain (such as C.H 1 and C L Antibody domain) or its variant. In some embodiments, TCRM is capable of recruiting at least one TCR-related signaling molecule selected from the group consisting of CD3δε, CD3γε and ζζ. In some embodiments, TCRM allows the recruitment of at least one TCR-related signaling molecule to be enhanced compared to TCRM comprising a T cell receptor transmembrane domain. In some embodiments, TCRM promotes the formation of caTCR-CD3 complexes. 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 portion (e.g., and an anti-GPC3 antibody portion). In some embodiments, the antibody portion is Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv).

[0095] In some embodiments, the anti-GPC3 caTCR described herein specifically binds to GPC3 and comprises: a) a first TCRD comprising a first TCR-TM derived from one of the transmembrane domains of a naturally occurring γδTCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring γδTCR, wherein the first and second TCRDs form a TCRM 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 connected to the first and / or second TCRD. In some embodiments, both TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first TCRD further comprises a first TCR 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 part of a connecting peptide of a TCR subunit from which the first TCR-TM is derived, or a variant thereof, and / or the second connecting peptide comprises all or part of a connecting peptide of a TCR subunit from which the second TCR-TM is derived, or a variant thereof. In some embodiments, the first and second connecting peptides are connected by a disulfide bond. In some embodiments, the first TCRD further includes the first TCR intracellular domain, and / or the second TCRD further includes the second TCR intracellular domain. In some embodiments, the first TCR intracellular domain includes the sequence of the intracellular domain of the TCR subunit derived from the first TCR-TM, and / or the second TCR intracellular domain includes the sequence of the intracellular domain of the TCR subunit derived from the second TCR-TM. In some embodiments, the first TCRD is a fragment of the TCR subunit derived from the first TCR-TM, and / or the second TCRD is a fragment of the TCR subunit derived from the second TCR-TM. In some embodiments, anti-GPC3 caTCR further includes at least one auxiliary intracellular domain comprising a T cell costimulatory signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30 or CD40). In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have a binding affinity for each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first stabilizing domain and the second stabilizing domain are connected by a disulfide bond. In some embodiments, the first stabilizing domain and the second stabilizing domain comprise antibody domains (such as C H 1 and C LAntibody domain) or its variant. In some embodiments, TCRM is capable of recruiting at least one TCR-related signaling molecule selected from the group consisting of CD3δε, CD3γε and ζζ. In some embodiments, TCRM allows the recruitment of at least one TCR-related signaling molecule to be enhanced compared to TCRM comprising a naturally occurring γδT cell receptor transmembrane domain. In some embodiments, TCRM promotes the formation of caTCR-CD3 complexes. 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 portion (e.g., and an anti-GPC3 antibody portion). In some embodiments, the antibody portion is Fab, Fab', (Fab')2, Fv or single-chain Fv (scFv).

[0096] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V 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 any one of about 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 any one of about 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 any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H , and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. L In some embodiments, the anti-GPC3caTCR comprises an antigen binding moiety comprising: a V 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. H , and a V comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR2 comprising the amino acid sequence of GDN, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 LIn some embodiments, the antigen binding moiety is a Fab.

[0097] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V 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 any one of about 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 any one of about 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 any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H , and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. L In some embodiments, the anti-GPC3caTCR comprises an antigen binding moiety comprising: a V 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. H , and a V 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 L In some embodiments, the antigen binding moiety is a Fab.

[0098] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V 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 any one of about 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 any one of about 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 any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H , and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. L In some embodiments, the anti-GPC3caTCR comprises an antigen binding moiety comprising: a V 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. H , and a V 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 L In some embodiments, the anti-GPC3 antibody portion is a Fab.

[0099] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24. H, and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:25 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 24; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 25 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising the amino acid sequence of SEQ ID NO: 24 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 25 L In some embodiments, the antigen binding moiety is a Fab.

[0100] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26. H , and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:27 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 26; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising the amino acid sequence of SEQ ID NO: 26 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the antigen binding moiety is a Fab.

[0101] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 28, or an amino acid sequence 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. H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:29 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 28; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 29 L In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising the amino acid sequence of SEQ ID NO: 28 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 29 L In some embodiments, the antigen binding moiety is a Fab.

[0102] In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a first TCRD and a second polypeptide chain comprising a second TCRD. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a V fused to a TCRD derived from a TCR alpha chain and a second polypeptide chain. H , the second polypeptide chain comprising a V fused to a TCRD derived from a TCRβ chain L In some embodiments, the anti-GPC3ca TCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a V fused to a TCRD derived from a TCR β chain. H , the second polypeptide chain comprising a V fused to a TCRD derived from a TCRα chain L In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a V fused to a TCRD derived from a TCR delta chain and a second polypeptide chain. H , the second polypeptide chain comprising a V fused to a TCRD derived from a TCRγ chain LIn some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a V fused to a TCRD derived from a TCRγ chain and a second polypeptide chain. H , the second polypeptide chain comprising a V fused to a TCRD derived from a TCRδ chain L As used herein, the term "fusion" means direct fusion or indirect fusion.

[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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 30; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 31. 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, 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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 10; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 5. 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, 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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 63; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 64. 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, 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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 65; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 66. 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, 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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 67; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 68. 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, 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 having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 69; and ii) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least about 80% (including, for example, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 70. 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, 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 encode a chimeric co-stimulatory receptor (CSR) that specifically binds to GPC3 (also referred to herein as an "anti-GPC3 CSR"). The anti-GPC3 CSR is capable of stimulating immune cells that functionally express the CSR on the surface after binding of a target ligand. The anti-GPC3 CSR comprises a ligand binding module that provides ligand binding specificity, a transmembrane module, and a co-stimulatory immune cell signaling module that allows stimulation of immune cells. 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 a ligand binding module, a transmembrane module, and a co-stimulatory 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 a ligand binding module, a transmembrane module, and a 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 polypeptide chain is covalently linked to the second polypeptide chain, such as by a peptide bond or by another chemical bond (such as a disulfide bond). In some embodiments, the first polypeptide chain is linked to the second polypeptide chain by at least one disulfide bond.

[0110] Examples of co-stimulatory immune cell signaling domains used in the anti-GPC3 CSRs of the present invention include: cytoplasmic sequences of co-receptors of T cell receptors (TCRs) that can cooperate with caTCRs (e.g., anti-GPC3 as described herein) to initiate signal transduction following caTCR engagement; and any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities.

[0111] In some cases, the signal generated by the TCR alone is insufficient to fully activate T cells, and a costimulatory signal is also required. Therefore, in some embodiments, T cell activation is mediated by two different categories of intracellular signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (referred to herein as "primary T cell signaling sequences") and sequences that act in an antigen-independent manner to provide secondary or costimulatory signals (referred to herein as "costimulatory T cell signaling sequences").

[0112] Primary immune cell signaling sequences that act in a stimulatory manner may contain a signaling motif, which is called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary immune cell signaling sequences containing ITAMs include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. A "functional" primary immune cell signaling sequence is a sequence that is capable of transducing immune cell activation signals when operably coupled to an appropriate receptor. A "non-functional" primary immune cell signaling sequence that may comprise a fragment or variant of a primary immune cell signaling sequence is unable to transduce immune cell activation signals. The anti-GPC3 CSRs described herein lack functional primary immune cell signaling sequences, such as functional signaling sequences comprising ITAMs. In some embodiments, the anti-GPC3 CSRs lack any primary immune cell signaling sequences.

[0113] The co-stimulatory immune cell signaling sequence can be part of the intracellular domain of a co-stimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0114] The targeting ligand of the anti-GPC3 CSR is GPC3. In some embodiments, the GPC3 is cell surface-bound GPC3. In some embodiments, the GPC3 targeting ligand is the same as the GPC3 target antigen of a caTCR (e.g., an anti-GPC3 caTCR as described herein) expressed in the same immune cell.

[0115] In some embodiments, the ligand binding moiety in the anti-GPC3 CSR is an antibody portion that specifically binds to GPC3 ("anti-GPC3 antibody portion"). In some embodiments, the antibody portion is Fab, Fab', (Fab')2, Fv, or single-chain Fv (scFv). In some embodiments, the ligand binding moiety is derived from the extracellular domain of the receptor. In some embodiments, the antibody portion of the anti-GPC3 CSR comprises the CDRs or variable domains (VDs) of an antibody portion that is specific for GPC3. H and / or V L domain).

[0116] In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H , and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8. H , and a V comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, LC-CDR2 comprising the amino acid sequence of GDN, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 L In some embodiments, the ligand binding moiety is a scFv.

[0117] In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a V-CDR 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 any one of about 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 any one of about 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 any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H, and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14. H , and a V 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 L In some embodiments, the ligand binding moiety is a scFv.

[0118] In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof comprising up to about 5 (such as any one of about 1, 2, 3, 4, or 5) amino acid substitutions. H , and a V 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 any one of about 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 any one of about 1, 2 or 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 any one of about 1, 2, 3, 4 or 5) amino acid substitutions. LIn some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20. H , and a V 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 L In some embodiments, the ligand binding moiety is a scFv.

[0119] In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24; H , and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:25 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 24; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 25 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising the amino acid sequence of SEQ ID NO: 24 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 25 L In some embodiments, the ligand binding moiety is a scFv.

[0120] In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 26, or an amino acid sequence 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; H, and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:27 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 26; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising the amino acid sequence of SEQ ID NO: 26 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the ligand binding moiety is a scFv.

[0121] In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a V comprising an amino acid sequence of SEQ ID NO: 28, or an amino acid sequence 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. H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 80% (including, for example, at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:29 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding moiety comprising: a) a V comprising the amino acid sequence of SEQ ID NO: 28; H ; and b) V comprising the amino acid sequence of SEQ ID NO: 29 L In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a V comprising the amino acid sequence of SEQ ID NO: 28 H HC-CDR, and V comprising the amino acid sequence of SEQ ID NO: 29 L In some embodiments, the ligand binding moiety 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, CD3ε, CD3ζ, 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 a 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 a 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) with SEQ ID NO: 50. In some embodiments, the anti-GPC3 CSR comprises a transmembrane domain derived from the CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or a 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) with SEQ ID NO: 22.

[0123] In some embodiments, the costimulatory immune cell signaling module of the anti-GPC3 CSR is derived from the intracellular domain of the costimulatory receptor of the TCR. In some embodiments, the costimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of: for example, all or part of the intracellular domain of a costimulatory receptor of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand specifically binding to CD83, and its analogs. In some embodiments, the costimulatory immune cell signaling module of the anti-GPC3 CSR comprises, consists essentially of, or consists of: all or part of the intracellular domain of a costimulatory receptor of CD30, CD28, 4-IBB, OX40, ICOS, CD27 or CD40. In some embodiments, the costimulatory immune cell signaling module of the CSR is derived from human CD30. In some embodiments, the costimulatory immune cell signaling module derived from human CD30 comprises the amino acid sequence of SEQ ID NO: 44, or a 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 costimulatory immune cell signaling module of CSR is derived from human CD28. In some embodiments, the costimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 45 or a 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) with SEQ ID NO: 45. In some embodiments, the costimulatory immune cell signaling module derived from human CD28 comprises the amino acid sequence of SEQ ID NO: 46 or a 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) with SEQ ID NO: 46. In some embodiments, the costimulatory immune cell signaling module of CSR is derived from human 4-IBB. In some embodiments, the costimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 47, or a 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) with SEQ ID NO: 47. In some embodiments, the costimulatory immune cell signaling module derived from human 4-IBB comprises the amino acid sequence of SEQ ID NO: 48, or a 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) with SEQ ID NO: 48.

[0125] In some embodiments, the anti-GPC3 CSR further comprises a spacer module between any two 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 one 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 moiety (such as an antibody portion) of the anti-GPC3 CSR specifically binds 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 greater) times greater than its binding affinity for other molecules; or b) a binding affinity that is at least about 10, 20, 30, 40, 50, 750, 1000, or greater than its binding affinity for other molecules. d Can exceed its K for binding to other molecules d The binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). d It can be determined by methods known in the art, such as surface plasmon resonance (SPR) using, for example, a Biacore instrument, or kinetic exclusion analysis (KinExA) using, for example, a Sapidyne instrument.

[0127] In some embodiments, the anti-GPC3 CSR described herein comprises a) a scFv; and b) a CD30 fragment. In some embodiments, the scFv comprises a V having an 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. H A V domain having an 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. L In some embodiments, the scFv comprises a V domain having an amino acid sequence of SEQ ID NO: 26. H domain and a V domain having an amino acid sequence of SEQ ID NO: 27 LIn some embodiments, the scFv comprises, consists essentially of, or consists of an 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 an amino acid sequence of SEQ ID NO: 33. In some embodiments, the fragment of CD30 comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, the CSR comprises, consists essentially of, or consists of an 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. Cc-Jun

[0128] The expression constructs provided herein further encode a c-Jun polypeptide.

[0129] In some embodiments, c-Jun is human c-Jun. In some embodiments, human c-Jun is wild-type human c-Jun. In some embodiments, human c-Jun has the following amino acid sequence (available at GenBank with accession number AAA59197.1 or at UniProtKB with accession number P05412.2): See also Hattori et al., PNAS (1988) 85:9148-52.

[0130] In humans, the c-Jun protein is encoded by the JUN gene located on chromosome 1 (GenBank Accession No. NC_000001.11, nucleotides 58,780,791 to 58,784,047, minus strand orientation). Synonyms for the JUN gene and its encoded protein 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 "enhancer binding protein AP1". The wild-type human c-Jun protein sequence is 331 amino acids in length.

[0131] In some embodiments, wild-type human c-Jun has 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, wild-type human c-Jun comprises the amino acid sequence of SEQ ID NO: 1.

[0132] In some embodiments, c-Jun is a mutant c-Jun. In some embodiments, c-Jun is a mutant human c-Jun. In some embodiments, the mutant c-Jun does not affect the ability of the mutant to rescue dysfunctional (exhausted) T cells. In some embodiments, the mutant c-Jun has at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95% or 99%) sequence identity with the C-terminal amino acid residue (e.g., C-terminal 50, 75, 100, 150, 200, 250 or more amino acid residues), C-terminal portion (e.g., one quarter, one third or one half) or C-terminal domain (e.g., its ε, bZIP and amino acid C-terminal) of wild-type c-Jun. In some embodiments, the N-terminal amino acid residues (e.g., the N-terminal 50, 75, 100, or 150 or more), N-terminal portion (e.g., one quarter, one third, or one half), or N-terminal domain (e.g., its delta, transactivation domain, and amino acids N-terminal) of wild-type c-Jun are deleted, mutated, or otherwise inactivated.

[0133] In some embodiments, c-Jun comprises one or more inactivating mutations (e.g., substitutions, deletions, or insertions) in its transactivation domain. In some embodiments, c-Jun comprises one or more inactivating substitutions in its transactivation domain. In some embodiments, c-Jun comprises one or more inactivating deletions in its transactivation domain. In some embodiments, c-Jun comprises one or more inactivating insertions in its transactivation domain. In some embodiments, c-Jun comprises one or more inactivating mutations (e.g., substitutions, deletions, or insertions) in its delta domain. In some embodiments, c-Jun comprises one or more inactivating substitutions in its delta domain. In some embodiments, c-Jun comprises one or more inactivating deletions in its delta domain. In some embodiments, c-Jun comprises one or more inactivating insertions in its delta domain. In some embodiments, c-Jun comprises one or more inactivating mutations (e.g., substitutions, deletions, or insertions) in its transactivation domain and its delta domain.

[0134] In some embodiments, c-Jun comprises a serine (S) to alanine (A) substitution. In some embodiments, c-Jun comprises one or both of the S63A and S73A substitutions compared to SEQ ID NO: 1 (positions are boxed in the amino acid sequence of human c-Jun above). In some embodiments, c-Jun has a deletion between amino acid residue 2 and amino acid residue 102 or between residues 30 and 50 compared to SEQ ID NO: 1.

[0135] In certain embodiments, the anti-GPC3 caTCR and / or anti-GPC3 CSR and / or c-Jun polypeptide further comprises an affinity or purification tag that can be used for various purposes; for example, it can 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, the immune cells described herein are modified to include 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 of the nucleic acid sequences listed in SEQ ID NOs: 52-62. In some aspects, the exogenous polynucleotide encoding a c-Jun polypeptide includes a nucleic acid sequence listed in any of SEQ ID NOs: 52-62.

[0137] In some aspects, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the exogenous polynucleotide encoding a c-Jun polypeptide has at least 96%, at least 97%, at least 98%, or at least 99% identity 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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] Any method known in the art can be used to make the c-Jun nucleotide sequence disclosed herein codon optimized. For example, in some aspects, the codons of the c-Jun nucleotide sequence disclosed herein have been optimized to modify (e.g., increase or decrease) one or more of the following parameters relative to the wild-type nucleotide sequence (e.g., SEQ ID NO: 62): (i) codon adaptation index (i.e., codon usage bias); (ii) guanine-cytosine (GC) nucleotide content; (iii) mRNA secondary structure and unstable motifs; (iv) repeat sequences (e.g., direct repeat sequences, inverted repeat sequences, binary repeat sequences (dyadrepeat)); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

[0149] Although certain disclosures provided herein generally relate to modifying immune cells to include exogenous nucleotide sequences encoding c-Jun proteins (wild-type c-Jun or variants 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 (wild-type or variants thereof) in cells. For example, as described herein, in some aspects, endogenous c-Jun protein expression can be increased using transcriptional activators (e.g., CRISPRa). Unless otherwise indicated, the disclosures provided above using exogenous nucleotide sequences are also applicable to other methods (e.g., transcriptional activators, such as CRISPRa) for inducing and / or increasing c-Jun protein expression in cells provided herein.

[0150] In some aspects, due to the modification (e.g., introduction of an exogenously introduced c-Jun nucleotide sequence and / or a transcriptional activator), the engineered cell overexpresses, i.e., expresses a higher level of c-Jun protein (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher, or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold higher) than a corresponding cell (a "reference cell") that does not contain such modification. The terms "expressing an increased level [or amount]," "overexpressing," or having "increased expression" (and similar forms of the phrases used herein) are used interchangeably.

[0151] In some aspects, the engineered (or modified) cells described herein express at least about 2-100 times more c-Jun protein than a reference cell, about 5-50 times more, about 5-40 times more, about 5-30 times more, about 5-20 times more, about 8-20 times more, or 10-20 times more. In some aspects, the expression of c-Jun protein in the modified cells described herein is increased by at least about 0.5 times, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12 times, at least about 14 times, at least about 16 times, at least about 18 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times compared to the expression of c-Jun protein in a reference cell.

[0152] In some aspects, increased c-Jun protein expression can improve and / or enhance one or more characteristics of modified immune cells (e.g., T cells, such as CD4+ and / or CD8+T cells). Non-limiting examples of such characteristics include: exhaustion resistance (e.g., indicated by exhaustion markers, such as PD-1, CD39, TIM-3 and / or LAG-3 reduced expression; increased persistence / survival rate; delayed onset of dysfunctional state; and / or increased cytokine (e.g., IFN-γ and / or IL-2) production); increased amplification / proliferation; increased antigen sensitivity; improved effector function, especially improved effector function after repeated antigen stimulation (e.g., producing cytokines after antigen stimulation, expressing cracking of target antigens, or both); or a combination thereof.

[0153] Assays suitable 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), Immune cell killing analysis, Meso Scale Discovery (MSD, a platform that combines electrochemiluminescence with MULTI-ARRAY technology) or similar analysis, persistent antigen stimulation analysis, 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 analysis, ELISA, Western blot and other standard molecular and cell biology methods (such as described herein or, for example, in Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021)) or other analysis. D. Nucleic acid

[0154] Anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun can be introduced into T cells or precursor cells via one or more nucleic acid molecules (e.g., DNA or RNA, such as mRNA). In some embodiments, the nucleic acid molecules can be placed on one or more DNA or RNA vectors for introduction into host cells.

[0155] Nucleic acid molecules (e.g., DNA or RNA vectors containing them) can be introduced into cells by well-known techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecular complexes, nanocapsules, microspheres and beads) and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles and liposomes). Alternatively, nucleic acid molecules can be introduced into cells by transduction of recombinant viruses, the genome of which comprises nucleic acid molecules. Examples of viral vectors include, but are not limited to, vectors derived from lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, Sendai viruses, and vaccinia viruses.

[0156] In some embodiments, the coding sequences for the two polypeptide chains of anti-GPC3 caTCR, anti-GPC3 CSR, and c-Jun can be placed on a single expression construct. The four coding sequences can be placed in one or more expression cassettes on the construct, each expression cassette being its own transcription unit (e.g., with its own promoter and polyadenylation site and other transcriptional control components). In a specific embodiment, the four coding sequences can be placed in a single expression cassette (e.g., a polycistronic expression cassette), wherein the four coding sequences are transcribed under a common promoter. In a polycistronic arrangement, the coding sequences are in the same frame and separated from each other by coding sequences 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 can be separated from each other by an internal ribosome entry site (IRES). Thus, the polycistronic (e.g., tetracistronic) expression cassette is transcribed into a single RNA, but ultimately the single RNA is processed and translated into independent polypeptides.

[0157] In some embodiments, the multicistronic expression cassette comprises the coding sequence of anti-GPC3caTCR, anti-GPC3 CSR and c-Jun. In some embodiments, the expression cassette comprises the coding sequence of the antigen binding moiety of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 28 and 29. In some embodiments, the expression cassette comprises the coding sequence of the TCRM derived from the human γ / δTCR. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 4 and 5. In some embodiments, the expression cassette comprises the coding sequence of two polypeptide chains of the anti-GPC3 caTCR. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 30 and 31. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 10 and 5. In some embodiments, the expression cassette comprises the coding sequence of the ligand binding moiety of the anti-GPC3 CSR. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the expression cassette comprises a coding sequence of a partial sequence of human CD30 of anti-GPC3CSR. In some embodiments, the expression cassette comprises a coding sequence of the amino acid sequence of SEQ ID NO:43. In some embodiments, the expression cassette comprises a coding sequence of a human c-Jun polypeptide. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO:1. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO:1, a coding sequence of SEQ ID NO:28 and 29, and a coding sequence of SEQ ID NO:33. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO:1, a coding sequence of SEQ ID NO:30 and 31, and a coding sequence of SEQ ID NO:33. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO:1, a coding sequence of SEQ ID NO:10 and 5, and a coding sequence of SEQ ID NO:33. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO:1, a coding sequence of SEQ ID NO:28 and 29, and a coding sequence of SEQ ID NO:33 and 43. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 30 and 31, and the coding sequence of SEQ ID NO: 33 and 43. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequence of SEQ ID NO: 10 and 5, and the coding sequence of SEQ ID NO: 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 the coding sequence of SEQ ID NO:2.

[0158] The expression cassette (polycistronic or monocistronic) may comprise a promoter that is constitutively active in mammalian (e.g., human or human T) cells. Such promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, the elongation factor-1α (EF-1α) promoter, the MND promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Core or minimal promoters derived from the aforementioned promoters are also contemplated. Alternatively, the expression cassette may comprise an inducible promoter system. Exemplary inducible promoter systems include, but are not limited to, hormone-regulated components, synthetic ligand-regulated components, ionizing radiation-regulated components, tetracycline (Tet) systems (e.g., "Tet-Off" and "Tet-On" systems), and NFAT 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 cassette includes a signal peptide. In some embodiments, the signal peptide comprises a coding sequence of the amino acid sequence of SEQ ID NO: 2.

[0159] In some embodiments, the expression cassette also includes a Kozak sequence, a polyadenylation site, and other components that promote transcription and / or translation of the coding sequence. For example, the woodchuck hepatitis virus post-transcriptional response element (WPRE) or a variant thereof can be included in the 3' untranslated region of the expression cassette.

[0160] In an expression cassette, transcription / translation regulatory elements, such as a promoter, any enhancers and the like, are operably linked to the coding sequence so as to allow efficient expression of the coding sequence and efficient translation of the RNA transcript.

[0161] In certain embodiments, the present invention provides a single vector construct (e.g., a lentiviral vector) comprising a multicistronic expression cassette comprising a mammalian promoter, a c-Jun coding sequence, a coding sequence for two anti-GPC3caTCR chains, a coding sequence for anti-GPC3CSR, and a polyadenylation signal sequence. The coding sequences are connected by a nucleotide linker, which may be an IRES or a coding sequence for a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A, or a functional equivalent thereof). By way of example, Figure 1ASuch an expression cassette is depicted, wherein the promoter is the EF-1α promoter.

[0162] In certain embodiments, the expression cassette encodes a c-Jun comprising SEQ ID NO: 1, an anti-GPC3 caTCR comprising two polypeptide chains comprising SEQ ID NO: 30 and 31, respectively, and an anti-GPC3 CSR comprising SEQ ID NO: 33 (e.g., Figure 1A The construct 2 in ). The construct may be a recombinant lentiviral vector and may further comprise a central polypurine tract (cPPT) upstream of the EF-1α promoter and a WPRE sequence between the CSR coding sequence and the SV40 polyadenylation signal or other sequences that are effective for transduction and expression in mammalian cells.

[0163] The coding sequence in the expression cassette can be codon-optimized for optimal expression in the host cell of interest (eg, human cells).

[0164] Nucleic acid molecules encoding anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun can be integrated into the genome of the engineered cells or remain free. The integration can be targeted integration occurring via gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases and meganucleases).

[0165] Engineered cells can be enriched by positive selection techniques. For example, cells can be selected based on their ability to bind GPC3 in, for example, flow cytometry analysis. To confirm c-Jun expression, engineered T cells can be subjected to RT-PCR. Positive selection can result in caTCR in the cell population. + CSR + c-Jun + Cells are enriched, wherein 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 can be cryopreserved until use. E. Variants

[0166] In some embodiments, amino acid sequence variants of the anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun provided herein are contemplated. In some embodiments, amino acid sequence variants of the antigen binding moiety of the anti-GPC3 caTCR and / or the ligand binding moiety of the anti-GPC3 CSR are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of one or more polypeptides encoded by the expression construct (e.g., anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun). Anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun amino acid sequence variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of 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, eg, GPC3 binding.

[0167] In some embodiments, anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include hypervariable regions (HVRs) and framework regions (FRs).

[0168] Conservative substitutions are shown in Table 2 below. Table 2. Conservative substitutions. Original residue Exemplary substitutions Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0169] Amino acids can be divided into different groups based on common side chain properties: a. Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; and f. Aromatic: Trp, Tyr, Phe.

[0170] Non-conservative substitutions will result in exchanging a member of one of these classes for another class.

[0171] Exemplary substitution variants are affinity-matured antibody portions consisting of an antigen-binding moiety of an anti-GPC3 caTCR or a ligand-binding moiety of an anti-GPC CSR, which can be conveniently produced, for example, using affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated and the variant antibody portion is displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) can occur in HVRs, for example, to improve antibody portion affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or specificity determining residues (SDRs), where the resulting variant V is tested. H or V L Affinity maturation by construction of secondary libraries and reselection from secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)).

[0172] In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody partial variants with the desired affinity. Another method of introducing diversity involves an HVR-guided approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling to identify. CDR-H3 and CDR-L3 are often particularly targeted.

[0173] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the anti-GPC3 caTCR and / or anti-GPC3 CSR to bind to GPC3. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two or three amino acid substitutions.

[0174] A useful method for identifying residues or regions of an antibody (e.g., the antigen binding moiety of an anti-GPC3 caTCR or the portion of an antibody contained in an anti-GPC3 CSR) that can 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 residues of interest (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitutions. Alternatively or in addition, a crystal structure of the antigen-antibody complex can be determined to identify contact points between the anti-GPC3 caTCR and / or anti-GPC3 CSR and GPC3. Such contact residues and neighboring residues can be targeted or excluded as candidates for substitution. Variants can be screened to determine whether they exhibit the desired properties.

[0175] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibody moieties (e.g., antigen binding moieties of anti-GPC3 caTCRs and / or ligand binding moieties of anti-GPC3 CSRs) having an N-terminal methionyl residue. Other insertion variants of antibody moieties include fusions of the N-terminus or C-terminus of an antibody moiety with an enzyme (e.g., for antibody-directed enzyme prodrug therapy, ADEPT) or a polypeptide that extends the serum half-life of a construct (e.g., an anti-GPC3 caTCR and / or an anti-GPC3 CSR construct). Glycosylation variants

[0176] In some embodiments, the anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun provided herein are altered to increase or decrease the extent of glycosylation of the expression construct. Addition or deletion of glycosylation sites to the expression construct can be conveniently achieved by altering the amino acid sequence of the anti-GPC3 caTCR, anti-GPC3 CSR and / or c-Jun construct or a polypeptide portion thereof to create or remove one or more glycosylation sites.

[0177] In the case where the anti-GPC3 caTCR comprises an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide generally linked to Asn297 of the CH2 domain of the Fc region by an N-bond (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-GPC3 constructs of the invention may be modified to produce anti-GPC3 construct variants with improved properties.

[0178] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 2006, 140: 777-83. Typically, a small percentage of naturally occurring defucosylated IgG can be detected in human serum. N-glycosylation of Fc is essential for binding to FcγR; and defucosylation of N-glycans increases the binding capacity of Fc to FcγRIIIa. Enhanced FcγRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0179] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. 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 generate 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 are present at accessible sites of the constructs described herein. By replacing these residues with cysteine, reactive thiol groups are located at accessible sites of the construct and can be used to bind the anti-GPC3 construct to other moieties. Cysteine ​​engineered expression constructs can be generated as described, for example, in U.S. Patent No. 7,521,541. III. Immune cells

[0181] In one aspect, the invention provides modified cells (such as immune cells, e.g., T cells) expressing anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptides (such as any of the anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptides described herein). Exemplary methods of preparing modified cells (such as T cells) expressing anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptides (such as any of the anti-GPC3 caTCR, anti-GPC3 CSR, and human c-Jun polypeptides 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, CSR and c-Jun polypeptide according to any one of the caTCR, CSR and c-Jun polypeptides described herein (such immune cells are also referred to herein as "caTCR plus CSR plus c-Jun immune cells"). The caTCR and CSR of the present invention specifically target GPC3 (such caTCR and CSR are also referred to herein as "anti-GPC3 caTCR" and "anti-GPC3 CSR", respectively). In some embodiments, the immune cell comprises a nucleic acid encoding an anti-GPC3 caTCR and an anti-GPC3 CSR, wherein the anti-GPC3 caTCR and anti-GPC3 CSR are expressed by the nucleic acid and localized on the surface of the immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells and suppressor T cells. In some embodiments, the immune cell does not express the TCR subunit from which the TCR-TM of the anti-GPC3 caTCR is derived. For example, in some embodiments, the immune cell is an αβT cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises a sequence derived from the TCRδ and γ chains, or the T cell is a γδT cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises a sequence derived from the TCRα and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or two of the endogenous TCR subunits of the immune cell. For example, in some embodiments, the immune cell is an αβT cell modified to block or reduce the expression of TCRα and / or β chains, or the immune cell is a γδT cell modified to block or reduce the expression of TCRγ and / or δ chains. Cell modifications for disrupting gene expression include any such techniques known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., gene knockout based on CRISPR or TALEN), and the like.

[0183] For example, in some embodiments, an immune cell (such as a T cell) is provided, comprising a 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, the anti-GPC3 CSR, and the human c-Jun polypeptide are expressed from the nucleic acid and localized on the surface of the immune cell. 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 anti-GPC3 caTCR nucleic acid sequence and the second anti-GPC3 CSR nucleic acid sequence and the human c-Jun nucleic acid sequence are each contained in different vectors. In some embodiments, some or all of the nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, a group consisting of a mammalian expression vector and a viral vector (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses). In some embodiments, one or more vectors are integrated into the host genome of the immune cell. In some embodiments, the first anti-GPC3 caTCR nucleic acid sequence and the second 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 cells are selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

[0184] Thus, in some embodiments, an anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun ("caTCR+CSR+c-Jun") immune cell (such as a T cell) is provided, which expresses 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 The caTCR polypeptide chain is expressed from the first and second anti-GPC3 caTCR nucleic acid sequences to form an anti-GPC3 caTCR, wherein the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid to form an anti-GPC3 CSR, wherein the c-Jun polypeptide is expressed from the c-Jun nucleic acid to form a c-Jun polypeptide, and wherein the anti-GPC3 caTCR and the anti-GPC3 CSR are localized on the surface of an 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 sequence is contained in a fourth vector (such as a lentiviral vector). In some embodiments, some or all of the first anti-GPC3 caTCR nucleic acid sequence and the second 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 sequences, and c-Jun nucleic acid sequences are contained in the same vector (such as a lentiviral vector). In some embodiments, the first and second anti-GPC3 caTCR nucleic acid sequences, anti-GPC3 CSR nucleic acid sequences, and c-Jun nucleic acid sequences are each 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 subunit of the TCR-TM derived from the caTCR. For example, in some embodiments, the immune cell is an αβT cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises a sequence derived from the TCRδ and γ chains, or the immune cell is a γδT cell and the TCR-TM of the introduced anti-GPC3caTCR comprises a sequence derived from the TCRα and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or two of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβT cell modified to block or reduce the expression of TCRα and / or β chains, or the immune cell is a γδT cell modified to block or reduce the expression of TCRγ and / or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors) that integrate into the host genome of the immune cell.

[0185] In some embodiments, a caTCR+CSR+c-Jun immune cell (such as a T cell) is provided, which expresses on its surface an anti-GPC3 caTCR according to any one of the caTCRs described herein, an anti-GPC3 CSR according to any one of the CSRs described herein, and a human c-Jun polypeptide according to any one 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 caTCR nucleic acid sequence encoding an anti-GPC3 caTCR polypeptide chain of the anti-GPC3 CSR; CSR nucleic acid sequence; and d) a fourth 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 an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence to form a c-Jun polypeptide, and wherein the caTCR and CSR are localized on the surface of immune cells. 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 cells do not express the TCR subunit of the TCR-TM from which the caTCR is derived. For example, in some embodiments, the immune cell is an αβT cell and the TCR-TM of the introduced caTCR comprises a sequence derived from TCRδ and γ chain, or the immune cell is a γδT cell and the TCR-TM of the introduced caTCR comprises a sequence derived from TCRα and β chain. In some embodiments, the immune cell is modified to block or reduce the expression of one or two of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβT cell modified to block or reduce the expression of TCRα and / or β chain, or the immune cell is a γδT cell modified to block or reduce the expression of TCRγ and / or δ chain. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) that integrate into the host genome of the immune cell.

[0186] In some embodiments, a caTCR+CSR+c-Jun immune cell (such as a T cell) is provided, which expresses on its surface an anti-GPC3 caTCR according to any one of the caTCRs described herein, an anti-GPC3 CSR according to any one of the CSRs described herein, and a c-Jun polypeptide according to any one 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 caTCR nucleic acid sequence encoding an anti-GPC3 caTCR polypeptide chain of the anti-GPC3 CSR; CSR nucleic acid sequence; 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 an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR is localized on the surface of an immune cell. In some embodiments, some or all of the promoters have the same sequence. In some embodiments, some or all of the promoters have different sequences. In some embodiments, some or all of the promoters are inducible. In some embodiments, the immune cell does not express the TCR subunit from which the TCR-TM of the anti-GPC3 caTCR is derived. For example, in some embodiments, the immune cell is an αβT cell and the TCR-TM of the introduced caTCR comprises a sequence derived from the TCRδ and γ chains, or the immune cell is a γδT cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises a sequence derived from the TCRα and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or two of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβT cell modified to block or reduce the expression of the TCRα and / or β chain, or the immune cell is a γδT cell modified to block or reduce the expression of the TCRγ and / or δ chain. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.In some embodiments, the first and second vectors are viral vectors (such as lentiviral vectors) that integrate into the host genome of the immune cell. It should be understood that embodiments in which any one of the nucleic acid sequences is exchanged are also contemplated, such as where the first or second anti-GPC3 caTCR nucleic acid sequence is exchanged with an anti-GPC3 CSR nucleic acid sequence or a c-Jun nucleic acid sequence.

[0187] In some embodiments, a caTCR+CSR+c-Jun immune cell (such as a T cell) is provided, which expresses on its surface an anti-GPC3 caTCR according to any one of the caTCRs described herein, an anti-GPC3 CSR according to any one of the CSRs described herein, and a c-Jun polypeptide according to any one 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 the anti-GPC3 caTCR nucleic acid sequence encoding the anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; CSR nucleic acid sequence; 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 an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed from the anti-GPC3CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR and the anti-GPC3 CSR are localized on the surface of immune cells. In some embodiments, a first promoter is operably linked to the 5' end of the first anti-GPC3 caTCR nucleic acid sequence, and a nucleic acid linker selected from the group consisting of an internal ribosome entry site (IRES) and a nucleic acid encoding a self-cleavage 2A peptide (such as P2A, T2A, E2A or F2A) is present to connect the 3' end of the 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 into a single RNA under the control of the promoter. In some embodiments, a first promoter is operably linked to the 5' end of the second anti-GPC3 caTCR nucleic acid sequence, and a nucleic acid linker selected from the group consisting of an internal ribosome entry site (IRES) and a nucleic acid encoding a self-cleavage 2A peptide (such as P2A, T2A, E2A or F2A) is present to connect the 3' end of the 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 into 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 cells do not express the TCR subunits of the TCR-TM derived from the caTCR. For example, in some embodiments, the immune cells are αβT cells and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from TCRδ and γ chains, or the immune cells are γδT cells and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from TCRα and β chains. In some embodiments, the immune cells are modified to block or reduce the expression of one or two of their endogenous TCR subunits. For example, in some embodiments, the immune cells are αβT cells modified to block or reduce the expression of TCRα and / or β chains, or the immune cells are γδT cells modified to block or reduce the expression of TCRγ and / or δ chains. In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the vector is a viral vector (such as a lentiviral vector) that integrates into the host genome of the immune cell. It should be understood that embodiments in which any one of the nucleic acid sequences is exchanged are also contemplated, such as where the first or second anti-GPC3 caTCR nucleic acid sequence is exchanged with an anti-GPC3 CSR nucleic acid sequence or a c-Jun nucleic acid sequence.

[0188] In some embodiments, a caTCR+CSR+c-Jun immune cell (such as a T cell) is provided, which expresses on its surface an anti-GPC3 caTCR according to any one of the caTCRs described herein, an anti-GPC3 CSR according to any one of the CSRs described herein, and a c-Jun polypeptide according to any one 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 polypeptide, wherein the first and second anti-GPC3 caTCR nucleic acid sequences, the anti-GPC3 The CSR nucleic acid sequence and 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 an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chains are expressed from the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed from the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR and the anti-GPC3 CSR are localized on the surface of immune cells. In some embodiments, the promoter is operably linked to a nucleic acid sequence that is linked to other nucleic acid sequences via a nucleic acid linker individually selected from the group consisting of an internal ribosome entry site (IRES) and a nucleic acid encoding a self-cleaving 2A peptide (such as P2A, T2A, E2A, or F2A), such that the first and second caTCR nucleic acid sequences and the CSR nucleic acid sequence are transcribed into a single RNA under the control of the promoter. In some embodiments, the promoter is inducible. In some embodiments, the immune cell does not express the TCR subunit from which the TCR-TM of the anti-GPC3caTCR is derived. For example, in some embodiments, the immune cell is an αβT cell and the introduced TCR-TM of the anti-GPC3 caTCR comprises a sequence derived from the TCRδ and γ chains, or the immune cell is a γδT cell and the introduced TCR-TM of the caTCR comprises a sequence derived from the TCRα and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or two of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβT cell modified to block or reduce the expression of the TCRα and / or β chain, or the immune cell is a γδT cell modified to block or reduce the expression of the TCRγ and / or δ chain.In some embodiments, the immune cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. In some embodiments, the vector is a viral vector (such as a lentiviral vector) that integrates into the host genome of the immune cell. A. Source of immune cells

[0189] The source of the engineered immune cells of the present disclosure can be the patient to be treated (i.e., autologous cells) or from a donor other than 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 can be CD4 + CD8 - (i.e. CD4 single positive) T cells, CD4 - CD8 + (i.e. CD8 single positive) T cells or CD4 + CD8 + (Double positive) T cells. Functionally, T cells can be cytotoxic T cells, helper T cells, natural killer T cells, suppressor T cells or mixtures thereof. The T cells to be engineered can be autologous or allogeneic.

[0190] Before T cell amplification and gene modification, T cell source is obtained from the subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In some embodiments of the present invention, any number of T cell lines available in the art can be used. In some embodiments of the present invention, T cells can be obtained from any number of techniques known to those skilled in the art (such as Ficoll TMA blood unit collected from a subject (separation). In some embodiments, cells from the circulating blood of an individual are obtained by hemopheresis. The hemopheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, the cells collected by hemopheresis can be washed to remove the plasma portion and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and may lack magnesium or may lack many (if not all) divalent cations. As will be readily understood by those skilled in the art, the washing step can be achieved by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics cell preserver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free. 2+ , Mg-free 2+ Alternatively, undesirable components of the hemopheresis sample can be removed and the cells resuspended directly in culture medium.

[0191] In some embodiments, T cells are separated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes, for example by PERCOLL TM Gradient centrifugation or counterflow centrifugal elutriation. Specific subsets of T cells (such as CD3 + 、CD28 + 、CD4 + 、CD8 + 、CD45RA + CD45RO + T cells) can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by binding to anti-CD3 / anti-CD28 (i.e., 3×28) beads, such as M-450CD3 / CD28 T cells are cultured together and isolated. In some embodiments, the time period is about 30 minutes. In some embodiments, the range of the time period is 30 minutes to 36 hours or longer and all integer values ​​therebetween. In some embodiments, the time period is at least 1, 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. In order to isolate T cells from leukemia patients, using a longer incubation time (such as 24 hours) can increase cell yield. In any case where T cells are rare compared to other cell types, a longer incubation time can be used to isolate T cells, such as isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. In addition, using a longer incubation time can increase the capture of CD8 + The efficiency of T cells. Therefore, by only shortening or extending the time allowing T cells to bind CD3 / CD28 beads and / or by increasing or reducing the ratio of beads to T cells, T cell subpopulations can be preferentially selected at the start of culture or at other time points during the process. In addition, by increasing or reducing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subpopulations can be preferentially selected at the start of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some embodiments, it may be necessary to perform a selection procedure and use "unselected" cells during activation and amplification. "Unselected" cells can also be subjected to other multiple rounds of selection.

[0192] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. One method is to sort and / or select cells via negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD-14, CD20, CD11b, CD-16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich or positively select cells that normally express CD4+. + 、CD25 + 、CD62Lhi、GITR + and FoxP3 + Alternatively, in some embodiments, T regulatory cells are depleted by anti-CD25-conjugated beads or other similar selection methods.

[0193] In order to separate the desired cell population by positive selection or negative selection, cell concentration and surface (e.g., particles, such as beads) can vary. In some embodiments, it may be necessary to significantly reduce the volume (i.e., increase cell concentration) in which beads and cells are mixed together to ensure the maximum contact of cells with 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 cell concentration of any one of about 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml is used. In some embodiments, a cell concentration of any one of about 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In some embodiments, a concentration of about 125 million or about 150 million cells / ml is used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. In addition, using high cell concentrations can more effectively capture cells that may weakly express the target antigen of interest, such as CD28 negative T cells, or cells from samples where there are many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and need to be obtained. For example, using high cell concentrations allows for more effective selection of CD8 T cells that typically have weak CD28 expression. + T cells.

[0194] In some embodiments of the present invention, T cells are obtained directly from patients after treatment. In this regard, it has been observed that after certain cancer treatments, especially after treatment with drugs that destroy the immune system, the quality of the T cells obtained can be optimal or their ability to be expanded in vitro is improved shortly after treatment during the period when the patient will usually recover from treatment. Similarly, after being manipulated in vitro using the methods described herein, these cells can be in a preferred state, thereby enhancing transplantation and in vivo expansion. Therefore, blood cells are collected during this recovery phase, including T cells, dendritic cells, or other cells of the hematopoietic lineage, within the context of the present invention. In addition, in some embodiments, movement (e.g., movement with GM-CSF) and conditioning therapy can be used to establish conditions within the subject, which are conducive to reconstruction, recirculation, regeneration and / or expansion of specific cell types, especially during a limited time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0195] Whether before or after genetically modifying T cells to express a desired expression construct, T cells can generally be activated and expanded using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0196] In general, the T cell lines of the present invention are expanded by contacting agents that stimulate CD3 / TCR complex-related signals and ligands that stimulate co-stimulatory molecules on the surface of T cells with the surface to which they are connected. Specifically, the T cell population can be stimulated as follows: such as by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD3 antibody fixed on the surface, or by contact with a protein kinase C activating factor (such as bryostatin) that binds to a calcium ion carrier. In order to costimulate auxiliary molecules on the surface of T cells, ligands that bind to auxiliary molecules are used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. In order to stimulate CD4 + T cells or CD8 + Anti-CD3 antibodies and anti-CD28 antibodies can be used to proliferate T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, France), and other methods generally known in the art may be used (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] Cell culture conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, reagents, such as nutrients, amino acids, antibiotics, ions and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors and any other reagents intended to activate cells. In some embodiments, the culture conditions include the addition of IL-2, IL-7 and / or IL-15.

[0198] In some embodiments, the cell to be engineered can be a multipotent or pluripotent cell that differentiates into a mature T cell after engineering. These non-T cells can be allogeneic, and can be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem cells or precursor cells. For ease of description, multipotent and pluripotent cells are collectively referred to as "precursor cells" herein.

[0199] Where allogeneic cells are used, the allogeneic cells are preferably engineered to reduce graft-versus-host rejection (eg, by knocking out endogenous B2M and / or TRAC genes). B. Engineering of immune cells or precursor cells

[0200] In some aspects, the cellular engineering of immune cells comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow tumor-specific targeting (e.g., 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., polynucleotides encoding c-Jun polypeptides such that the immune cells exhibit increased c-Jun expression compared to corresponding cells that have not been modified), or any combination thereof. As further described elsewhere in this disclosure, in some aspects, cells can be engineered or modified with transcriptional activators (e.g., transcriptional activators based on a CRISPR / Cas system), wherein the transcriptional activators are capable of inducing and / or increasing endogenous expression of a protein of interest (e.g., c-Jun).

[0201] In some aspects, the cells described herein have been modified with transcriptional activators that can induce and / or increase the endogenous expression of a protein of interest (e.g., c-Jun) in a cell. As used herein, the term "transcriptional activator" refers to a protein that increases the transcription of a gene or genome (e.g., by binding to an enhancer or promoter proximal component of a nucleic acid sequence, thereby inducing its transcription). Non-limiting examples of such transcriptional activators that can be used with the present invention include: transcriptional activators based on transcriptional activator-like effectors (TALEs), transcriptional activators based on zinc finger proteins (ZFPs), transcriptional activators based on clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (Cas) systems, or combinations thereof. See, for example, Kabadi et al., Methods (2014) 69 (2): 188-197, which is incorporated herein by reference in its entirety.

[0202] In some aspects, the cells described herein have been modified with a transcriptional activator based on a CRISPR / Cas system, 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 includes the use of a modified Cas protein that lacks endonuclease activity but retains the ability to bind to its guide RNA and target DNA nucleic acid sequence. Non-limiting examples of such modified Cas proteins that can be used in the present invention are known in the art (see, for example, 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 as "dCas9" in the art). In some aspects, the modified Cas protein comprises a modified Cas12a protein. In some aspects, the modified Cas protein suitable for the present invention binds to a guide polynucleotide (e.g., a small guide RNA) ("modified Cas guide complex"), wherein the guide polynucleotide comprises a recognition sequence complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In some aspects, the guide polynucleotide comprises a recognition sequence complementary to a 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-terminus 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 transcriptional activators can bind to the regulatory elements (e.g., promoter regions) of the endogenous gene, thereby inducing and / or increasing the expression of the encoded protein (e.g., c-Jun). Illustrative examples of common general activators that can be used include RNAP, VP16, VP64, and the ω subunit of 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)) may be attached to a modified Cas guide complex (e.g., the N-terminus and / or C-terminus of a modified Cas protein) such that when introduced into a cell, the one or more transcriptional repressors may inhibit or reduce transcription of genes, such as those that interfere with c-Jun expression (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 suitable for use in the present invention may be attached to both one or more transcriptional activators and one or more transcriptional repressors.

[0204] Without being bound by any one theory, in some aspects, the use of such modified Cas proteins can allow conditional transcription and expression of genes of interest. For example, in some aspects, cells (e.g., T cells) are modified to include a recombinant antigen receptor (e.g., anti-GPC3 caTCR and anti-GPC3 CSR) connected 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 include a linker (LAT) for activating T cells, which is complexed with a modified Cas protein, which is attached to a transcriptional activator (e.g., dCas9-VP64-p65-Rta transcriptional activator (VPR)) via a linker (e.g., a TEV cleavable linker). After antigen receptor activation, 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(Suppl 2):A164, which is incorporated herein by reference in its entirety.

[0205] As will be apparent to one skilled in the art, in some aspects, the cells described herein have been modified using a combination of methods. For example, in some aspects, the cells have been modified to include (i) an exogenous nucleotide sequence encoding one or more proteins (e.g., anti-GPC3caTCR, anti-GPC3 CSR, and truncated EGFR (EGFRt)) and (ii) an exogenous transcriptional activator (e.g., CRISPRa) that increases the expression of an endogenous protein (e.g., c-Jun). In some aspects, the cells have been modified to include (i) an exogenous nucleotide sequence encoding a first protein (e.g., anti-GPC3caTCR), (ii) an exogenous nucleotide sequence encoding a second protein (e.g., anti-GPC3 CSR), and (iii) an exogenous nucleotide sequence encoding a protein (e.g., c-Jun protein). In some aspects, the modified cells may further include 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 may be part of a single polycistronic vector.

[0206] Unless otherwise indicated, one or more exogenous nucleotide sequences and / or transcriptional activators can be introduced into cells using any suitable method known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to cells include: transfection (also referred to as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof.

[0207] In some aspects, the cells have been modified with a transcriptional activator (e.g., a transcriptional activator based on a CRISPR / Cas system, such as CRISPRa) such that expression of endogenous c-Jun protein is increased compared to corresponding cells that have not been modified with the transcriptional activator.

[0208] Although certain disclosures provided herein are generally about modifying immune cells to include exogenous nucleotide sequences encoding c-Jun proteins (wild-type c-Jun or variants 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 (wild-type or variants thereof) in cells. For example, as described herein, in some aspects, endogenous c-Jun protein expression can be increased using transcriptional activators (e.g., CRISPRa). Unless otherwise indicated, the disclosures provided herein using exogenous nucleotide sequences are also applicable to other methods provided herein for inducing and / or increasing c-Jun protein expression in cells (e.g., transcriptional activators, such as CRISPRa).

[0209] The immune cells (e.g., T cells) or precursor cells herein can be engineered to express anti-GPC3 caTCR and anti-GPC3 CSR, and overexpress c-Jun (e.g., human c-Jun polypeptide). The anti-GPC3 caTCR can specifically bind to GPC3 tumor cells, and the anti-GPC3 CSR can specifically bind to GPC3 on tumor cells (e.g., the same GPC3, but different GPC3 epitopes). As used herein, when the bound K D A receptor (eg, an anti-GPC3 caTCR or an anti-GPC3 CSR) is said to specifically bind GPC3 when the K is ≤ 100 nM, such as ≤ 10 nM or ≤ 1 nM. D Binding affinity constants can be determined, for example, by surface plasmon resonance (using, for example, Biacore TM or Octet TM system) measurements.

[0210] In some embodiments, the immune cells express anti-GPC3 caTCRs with uniform cell surface distribution. In some embodiments, the immune cells express anti-GPC3 CSRs with uniform cell surface distribution. Uniform cell surface distribution can be characterized, for example, by a staining pattern having a continuous appearance and uniform thickness or signal intensity. For example, in some embodiments, a composition (such as a pharmaceutical composition) comprising anti-GPC3 caTCRs, anti-GPC3 CSRs, and human c-Jun immune cells comprises less than about 10% (such as less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of cells with anti-GPC3 caTCRs and / or anti-GPC3 CSRs aggregated on the cell surface. Aggregation can be characterized, for example, by a staining pattern having uneven thickness or signal intensity or a discontinuous, blocky, dotted, and / or uneven distribution pattern. In some embodiments, caTCR+CSR+c-Jun T cells express anti-GPC3 caTCR and / or anti-GPC3 CSR, wherein the amount of aggregation of anti-GPC3 caTCR and / or anti-GPC3 CSR on the cell surface is less than about 10% (such as less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%). In some embodiments, anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cells express anti-GPC3 caTCR and / or anti-GPC3 CSR, wherein the amount of aggregation of anti-GPC3 caTCR and / or anti-GPC3 CSR on the cell surface is less than about 10% (such as less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%). In some embodiments, anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cells have low levels of antigen-independent anti-GPC3 caTCR and / or anti-GPC3 CSR activation. In some embodiments, anti-GPC3 caTCR T cells have low levels of antigen-independent anti-GPC3 caTCR and / or anti-GPC3 CSR activation. In some embodiments, anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cells have low levels of T cell exhaustion. T cell exhaustion occurs naturally during prolonged immune activation conditions, such as in cancer or chronic infection situations, where T cells become dysfunctional. T cell exhaustion can be characterized by reduced effector function, prolonged expression of inhibitory receptors, and / or altered transcriptional states compared to functional effector or memory T cells. T cell exhaustion impedes optimal clearance of tumor cells and infections.T cell exhaustion of anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cells can be characterized by any means known in the art, such as by measuring their functional and / or phenotypic profiles (Wherry, EJ, Nature immunology 12(6):492-499, 2011; Jiang, Y. et al., Cell death & disease 6(6):e1792, 2015). For example, in some embodiments, anti-GPC3 caTCR plus anti-GPC3 CSR plus human c-Jun T cells express low levels of one or more T cell exhaustion markers, 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 cells maintain IL-2 production, TNF-α production, IFN-γ production, and granzyme B production characterized by non-exhausted T cells and / or maintain ex vivo killing capacity in the presence of target cells, indicating that caTCR+CSR+c-Jun T cells do not undergo 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 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 on the surface of the immune cell. In some embodiments, the caTCR+CSR+c-Jun immune cell composition is a pharmaceutical composition.

[0212] The composition may include: a homogeneous 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., wild-type or mutant c-Jun polypeptides). The composition may further include cells that are not caTCR+CSR+c-Jun immune cells.

[0213] At various points during the preparation of the composition, it may be necessary or desirable to cryopreserve the cells. The terms "frozen" or "freezing" and "cryopreserved" or "cryopreserving" are used interchangeably. Freezing includes freeze drying.

[0214] As will be appreciated by those of ordinary skill in the art, freezing cells can be damaging (see Mazur, P., 1977, Cryobiology 14:251-272), but there are a number of procedures available to prevent such damage. For example, damage can be avoided by (a) using a cryoprotectant, (b) controlling the freezing rate, and / or (c) storing at a sufficiently low temperature to minimize degradation reactions. Exemplary cryoprotectants include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), glycerol, polyvinyl pyrrolidone (Rinfret, 1960, Ann. NY Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 190:1204-1205), and polyvinyl pyrrolidone (Rinfret, 1960, Ann. NY Acad. Sci. 85:576). 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, monoacetin (Lovelock, 1954, Biochem. J. 56:265), and inorganic salts (Phan and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan and Bender, 1961, Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery ed., Butterworth, London, p. 59). In certain embodiments, DMSO may be used. Adding plasma (e.g., to a concentration of 20-25%) may enhance the protective effect of DMSO. After adding DMSO, cells may be kept at 0°C until frozen, as 1% DMSO concentrations may be toxic at temperatures above 4°C.

[0215] In cryopreservation of cells, a slow, controlled cooling rate can be critical and different cryoprotectants (Rapatz et al., 1968, Cryobiology 5(1):18-25) and different cell types have different optimal cooling rates (for the effect of cooling rate on the survival of stem cells and on their transplantation potential, 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). The heat of the melting phase of water into ice should be minimal. The cooling program can be performed using, for example, a programmable freezing device or a methanol bath program. The programmable freezing device allows the determination of the optimal cooling rate and facilitates standard, reproducible cooling.

[0216] In a particular embodiment, DMSO-treated cells can be pre-cooled on ice and transferred to a tray containing refrigerated methanol, which in turn is placed in a mechanical refrigerator (e.g., Harris or Revco) at -80°C. Thermocouple measurements of the methanol bath and samples indicate that a cooling rate of 1°C to 3°C / minute may be preferred. After at least two hours, the sample can reach a temperature of -80°C and can be placed directly in liquid nitrogen (-196°C).

[0217] After complete freezing, the cells can be quickly transferred to long-term cryogenic storage containers. In a preferred embodiment, samples can be cryogenically stored in liquid nitrogen (-196°C) or vapor (-1°C). The availability of high-efficiency liquid nitrogen freezers facilitates such storage.

[0218] Additional considerations and procedures for manipulation, cryopreservation, and long-term storage of cells can be found in the following exemplary references: U.S. Pat. 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. Parent. Sci. Technol. 46(6): 226-32.

[0219] After cryopreservation, the frozen cells can be thawed for use according to methods known to those of ordinary skill in the art. The frozen cells are preferably thawed quickly and refrigerated immediately after thawing. In a specific embodiment, the vial containing the frozen cells can be immersed in a warm water bath up to its neck; gentle rotation will ensure that the cell suspension is mixed as it thaws and improve heat transfer from the warm water to the internal ice. Once the ice is completely melted, the vial can be immediately placed on ice.

[0220] In certain embodiments, methods for preventing cell aggregation may be used during thawing. Exemplary methods include: adding deoxyribonuclease (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. before and / or after freezing. As will be appreciated by those of ordinary skill in the art, if a cryoprotectant toxic to humans is used, it should be removed prior to therapeutic use. DMSO is not severely toxic.

[0221] Exemplary carriers and modes of cell administration are described in U.S. Patent Publication No. 2010 / 0183564, pp. 14-15. Other pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).

[0222] In certain embodiments, cells can be collected from the culture medium, washed and concentrated in a carrier to a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0223] In certain embodiments, the carrier can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In certain embodiments, the infusion carrier includes buffered saline containing 5% HSA or dextrose. Other isotonic agents include polyhydroxy sugar alcohols, including triols or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0224] The carrier may include a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, and / or a trimethylamine salt.

[0225] Stabilizers refer to a broad class of excipients whose functionality can range from bulking agents to additives that help prevent cells from adhering to the vessel walls. Typical stabilizers may include polyhydroxy 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, fucose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol and cyclic alcohols such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran.

[0226] Where necessary or advantageous, the composition may include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0227] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexahydroxyquaternary ammonium chloride, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0228] The therapeutically effective amount of cells in the composition may 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 10 10 cells or more than 10 11 cells.

[0229] In the compositions and formulations disclosed herein, the volume of cells is generally one liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Thus, the cell density administered is generally greater than 10 4 cells / mL, 10 7 cells / mL or 10 8 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 the following: an isotonic agent, an excipient, a diluent, a thickener, a stabilizer, a buffer and / or a preservative; and / or an aqueous vehicle, such as purified water, a sugar solution, a buffer solution, a physiological saline solution, a polymer aqueous solution, or ribonuclease-free water. The amount of these additives and aqueous vehicles to be added can be appropriately 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 be further formulated for intramedullary, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular and / or subcutaneous injection.

[0232] Formulations to be used for in vivo administration must be sterile. This requirement is readily accomplished, for example, by filtration through sterile filtration membranes.

[0233] In some embodiments, the engineered immune cells can be collected from the culture medium, washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline (e.g., phosphate-buffered saline), normal saline, water, Hanks' solution, Ringer's solution, Nonnosol-R (AbbottLabs), Plasma-Lyte A (R) (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof. Preferably, the carrier is isotonic. In some embodiments, the carrier may be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose. Other isotonic agents may also be included, including polyhydroxy sugar alcohols, including trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0234] Medical immune cell compositions (such as medical T cell compositions) can be administered to cancer patients systemically (e.g., via intravenous or portal vein injection) or locally (e.g., via intratumoral injection) in a therapeutically effective amount. In some embodiments, compositions (such as compositions targeting GPC3) are used to treat patients with hepatocellular carcinoma (HCC), gastric cancer, pancreatic cancer, or reproductive system cancer (see, e.g., Wang and Wang, Canadian J Gastroent Hep. (2018) art. 9049252). As used herein, the term "treatment" or "treating" refers to a method for obtaining a beneficial or desired result in a treated subject. Such results include, but are not limited to, relieving one or more symptoms caused by a disease (e.g., HCC), reducing the scope of the disease (e.g., reducing tumor volume), stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread (e.g., metastasis), preventing or delaying disease recurrence or recurrence, improving the disease state, causing disease remission (partial or complete), reducing the dose of one or more other agents required to treat the disease, improving quality of life, restoring body weight, and / or prolonging survival (e.g., overall survival or progression-free survival).

[0235] A therapeutically effective amount of a composition refers to a 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 An engineered cell.

[0236] In some embodiments, the pharmaceutical composition comprises cells of an amount effective to treat or prevent a disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the therapeutic or prophylactic efficacy is monitored by regularly evaluating the treated subject. For repeated administration over several days or longer, depending on the condition, repeated treatment is performed until the desired inhibition of disease symptoms occurs. However, other dosing regimens are applicable and can be determined. The desired dose can be delivered by administering the composition by a single bolus, by administering the composition by multiple boluses, or by administering the composition by continuous infusion.

[0237] In certain embodiments, a subject is administered a range of about one million to about 100 billion cells, such as 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 bounded by any two of the above 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, or a range bounded by any two of the above values). 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 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 bounded by any two of the preceding 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 therebetween.

[0238] In some embodiments, cells and compositions are administered using standard administration techniques, formulations and / or devices. Formulations and devices for storing and administering compositions, such as syringes and vials, are provided. Administration may be autologous or allogeneic. For example, immunoreactive cells or precursor cells may be obtained from a subject and administered to the same subject or to different compatible subjects. Immunoreactive cells or their progeny (e.g., in vivo, ex vivo, or in vitro sources) derived from peripheral blood may 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 invention (e.g., a pharmaceutical composition containing genetically modified cells), it is typically formulated into a unit dose injectable form (solution, suspension, emulsion).

[0239] In one aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid molecule for expressing anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun. The nucleic acid molecule can be as described above, for example, a viral vector (e.g., a lentiviral vector) as described above. The pharmaceutical composition can be used in vitro to engineer T or precursor cells, which are then introduced into a patient. The pharmaceutical composition comprises a nucleic acid molecule or recombinant virus whose genome comprises an expression cassette for anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun and a pharmaceutically acceptable carrier, such as a buffer solution, which optionally contains other agents, such as preservatives, stabilizers and the like.

[0240] The pharmaceutical composition can be provided as an article of manufacture, such as a kit, which includes a vial (eg, a single-dose vial) containing the biological material (cells or nucleic acid molecules or recombinant viruses) and, optionally, instructions for use. V. Treatment Methods

[0241] The expression constructs and / or compositions (such as pharmaceutical compositions) of the invention can be administered to an individual (e.g., a mammal, such as a human) to treat diseases and / or disorders involving abnormally high expression of GPC3 (also referred to herein as "GPC3-positive" diseases or disorders), including, for example, cancer (such as hepatocellular carcinoma (HCC)).

[0242] Therefore, 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 one of the anti-GPC3 caTCRs provided herein. In some embodiments, the anti-GPC3 CSR is any one of the anti-GPC3 CSRs provided herein. In some embodiments, the c-Jun polypeptide is any one of the c-Jun polypeptides described herein.

[0243] In some embodiments, provided herein is a method of treating 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 caTCR, anti-GPC3 CSR, and c-Jun polypeptides described herein). In some embodiments, the cancer is selected from, for example, 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 cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, and liposarcoma. In some embodiments, the cancer is HCC. In some embodiments, the cancer is HCC and the treatment comprises preventing the spread of the cancer, for example, inhibiting (such as preventing) cancer metastasis. 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, the expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3caTCR comprising: i) an antigen binding moiety 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 A CSR comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to the immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the TCRM of the anti-GPC3 caTCR is derived from a human γ / δ TCR. In some embodiments, the anti-GPC3 caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the transmembrane module of the anti-GPC3 CSR comprises a transmembrane domain 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 wild-type human c-Jun polypeptide. In some embodiments, the cancer is selected from the group consisting of, for example, HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, lung squamous cell carcinoma, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, 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 the following: one or more expression constructs, the expression constructs comprising one or more expression cassettes for expressing the following: a) an anti-GPC3 caTCR comprising: i) an antigen binding moiety comprising: (a) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; H and (b) a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23. L ; 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding module comprising: (a) a V comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 H and (b) a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a LC-CDR2 comprising the amino acid sequence of YDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17 L ; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module capable of providing a co-stimulatory signal to the immune cell, wherein the ligand binding module and the co-stimulatory immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3caTCR comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L. 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 stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first stabilizing domain and the second stabilizing domain have binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the ligand binding module of the anti-GPC3 CSR comprises a V comprising the amino acid sequence of SEQ ID NO:26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L . 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 a transmembrane domain 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 wild-type 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 from the group consisting of, for example, HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, 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 antigen binding moiety 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 promotes recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding moiety comprising a scFv capable of binding or interacting with GPC3; ii) a transmembrane moiety derived from CD30; and iii) a costimulatory immune cell signaling moiety derived from CD30 capable of providing a costimulatory signal to an 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 (V H ) and a light chain variable domain (V L In some embodiments, the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26. H and V comprising the amino acid sequence of SEQ ID NO: 27 L 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 from the group consisting of, for example, HCC, melanoma, lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, triple negative breast cancer, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, 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 multicistronic expression construct, the multicistronic 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 a human γ / δ 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 the intracellular domain of human CD30; and c) a human c-Jun polypeptide. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequences of SEQ ID NO: 28 and 29, and the coding sequence of SEQ ID NO: 33. In some embodiments, the coding sequence is separated in frame by a 2A coding sequence or by an internal ribosome entry site (IRES). In some embodiments, the expression cassette comprises a constitutive or inducible promoter. In some embodiments, the promoter is an EF-1α promoter. In some embodiments, the construct is a viral vector (such as a lentiviral vector). In some embodiments, the cancer is selected from, for example, 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 cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer and liposarcoma.

[0248] In order to better understand the present invention, the following examples are described. These examples are for illustrative purposes only and should not be interpreted as limiting the scope of the present invention in any way. Example

[0249] The present invention will be more fully understood with reference to the following examples. However, it should not be construed as limiting the scope of the present invention. It should be understood that the embodiments and embodiments described herein are for illustrative purposes only, and various modifications or changes thereof will be proposed by those skilled in the art and are included within the spirit and scope of the present application and within the scope of the appended claims.

[0250] In the studies described in Examples 1-3 below, 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 expressing Glypican-3 (GPC3). 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, referred to herein as "GPC3-caTCR+GPC3-CD30-CSR" or "Construct 1," contains a tricistronic expression cassette encoding a GPC3-specific caTCR (SEQ ID NOs: 30 and 31) and a GPC3-specific CSR (i.e., GPC3-CD30-CSR) (SEQ ID NO: 16) containing a co-stimulatory domain derived from CD30. The second vector, referred to herein as "c-Jun+GPC3-caTCR+GPC3-CD30-CSR" or "Construct 2," contains a tetracistronic expression cassette encoding a GPC3-specific caTCR (SEQ ID NOs: 30 and 31), a GPC3-CD30-CSR (SEQ ID NO: 16), and human c-Jun (SEQ ID NO: 1). In each vector, the expression cassette is under the transcriptional control of the EF-1α promoter, and the coding sequences of the different polypeptides are cleaved in frame by a self-cleaving peptide (F2A, T2A or P2A, e.g. Figure 1A The coding sequence of the construct is connected to the lentiviral vector. The recombinant virus is produced by transfecting 293T cells with a vector encoding the construct using a known lentiviral production protocol and packaging system.

[0252] Primary human T cells were cultured with CD3 / CD28 beads ( Invitrogen) in the presence of 100 U / mL interleukin-2 (IL-2) for one day before lentiviral transduction. The concentrated lentivirus was applied to the T cells were plated in 6-well plates coated with PDMS (Takara) and incubated for 96 hours. Transduction efficiency was assessed by flow cytometry on day 9 using PE-conjugated anti-human F(ab')2 antibody. BD FACSCanto II TM Flow cytometry data were collected and analyzed using the FlowJo software package.

[0253] For in vivo studies, the antitumor activity of primary human T cells expressing construct 1 or construct 2 was tested in two liver cancer models, namely HepG2-luc (Study 1 and Study 2) and Hep3B model (Study 3). Luciferase-expressing HepG2 (HepG2-luc) cells or Hep3B cells were implanted subcutaneously (sc) into the right flank of NSG-MHC I / IIDKO mice (described in Figure 1B When the tumor size reaches about 150 mm 3 At 4 pm (20 days after tumor cell implantation), mice were injected intravenously (iv) with modified T cells.

[0254] The health effects of T cell infusion on mice were evaluated by monitoring the animals' systemic appearance, body weight, and other clinical signs of adverse reactions (including hypothermia, dyspnea, and hind limb paralysis / weakness). In addition, peripheral blood T cell counts were measured on day 14 after T cell administration. Example 1: Characterization of anti-GPC3 caTCR + anti-GPC3 CSR T cells overexpressing c-Jun in in vitro long-term target engagement assay

[0255] This example describes the effect of c-Jun overexpression on the cytotoxicity of human T cells engineered to express an anti-GPC3 caTCR and an anti-GPC3 CSR comprising a costimulatory domain derived from CD30 (i.e., GPC3-CD30-CSR). Specifically, this example demonstrates that c-Jun overexpression promotes the long-term target cell killing ability of T cells expressing anti-GPC3 caTCR+anti-GPC3 CSR. In terms of long-term T cell exhaustion, T cells expressing anti-GPC3 caTCR+anti-GPC3 CSR expressed comparably in the presence or absence of c-Jun overexpression.

[0256] Primary T cells isolated from healthy human donors were transduced with lentiviral construct 1 (i.e., GPC3-caTCR+GPC3-CD30-CSR) or construct 2 (i.e., c-Jun+GPC3-caTCR+GPC3-CD30-CSR) for 7-9 days. Tumor cytotoxicity was analyzed using a nonradioactive cytotoxicity assay (Promega) that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. CD3+ T cells were analyzed using EasySep TMHuman T cells were prepared from whole blood enriched for PBMC using the Human T Cell Isolation Kit (StemCell Technologies) which depletes cells expressing CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A. Human T cell lines were isolated using CD3 / CD28 (Invitrogen). Activated T cells (ATC) were cultured and maintained in RPMI-1640 medium with 10% FBS plus 100 U / mL IL*2 and used on days 7-14. Effector cells were expressed by caTCR based on human F(ab')2 staining (caTCR + ) Normalization (adjust all effector cell samples to the same percentage of receptor among total T cells using untransduced / mock-transduced T cells) + (caTCR + )cell).

[0257] T cells and target cells were co-cultured at a starting effector cell to target cell ratio (E:T ratio) of 1:1. Specifically, 50,000 caTCR + T cells were incubated with 50,000 HepG2 cells in RPMI-1640 and 10% FBS. To assess the long-term killing potential of T cells, the cells were re-challenged with 100,000 HepG2 cells per well every 7 days. After each target cell was engaged, the remaining target cells and caTCR were quantified. + The number of T cells.

[0258] Comparison of long-term killing (indicated by the amount of remaining target cells and the number of remaining T cells) is shown in Table 3. T cells expressing construct 2 cleared target cells via more rechallenge than T cells expressing construct 1, indicating that c-Jun promotes anti-GPC3 caTCR + Anti-GPC3 CSR + Long-term target cell killing capacity of T cells.

[0259] After engagement of each target cell, CD4 or CD8 single positive caTCRs were analyzed by flow cytometry. + The data in Table 3 show that c-Jun overexpression produces a higher percentage of CD4 + T cells but a lower percentage of CD8 + T cells, indicating that c-Jun expression promotes CD4 + T cells are preferentially expanded.

[0260] To determine whether transduction with construct 1 or construct 2 had an effect on T cell differentiation during long-term killing assays, CD8 + Receptors + The expression of CCR7 and CD45RA in the initial T cells. + CD45RA + Characterization, while memory T cells are characterized by CCR7 + CD45RA - As shown in Table 3, the expression level of CCR7 and the number of memory T cells and naive T cells were comparable between T cells expressing construct 1 and T cells expressing construct 2. These results indicate that c-Jun does not affect anti-GPC3 caTCR + Anti-GPC3 CSR + Differentiation of memory T cells.

[0261] To examine the levels of exhaustion markers expressed on T cells transduced with construct 1 or construct 2 during long-term killing assays, CD3 + T cells and stained with CD3 / CD28 Cells were activated. T cells were transduced with lentiviral construct 1 or construct 2 for 7-9 days. Effector cells were activated by caTCR based on human F(ab')2 staining. + Normalization. T cells were cultured as described above and re-challenged with HepG2 cells. caTCR was analyzed by flow cytometry on selected days after each target cell engagement + CD8 + T cells and caTCR + CD4 + Exhaustion markers PD-1, LAG-3 and TIM-3 on T cells PD-1, LAG-3, and TIM-3 are inhibitory receptors that accumulate on T cells as they lose function; therefore, these molecules are used as markers of T cell exhaustion.

[0262] As shown in Table 3, both groups of T cells showed similar levels of exhaustion markers. These results confirm that anti-GPC3 caTCR + Anti-GPC3 CSR + Expression was comparable in T cells in the presence or absence of c-Jun overexpression. Table 3. In vitro long-term target engagement results Example 2: In vivo efficacy of anti-GPC3caTCR+anti-GPC3CSR T cells overexpressing c-Jun in a re-challenge study in the HepG2-luc mouse model

[0263] This example demonstrates that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR and overexpressing c-Jun increase tumor killing in vivo compared to T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.

[0264] As described above, primary human T cells from two different donors expressing construct 1 or construct 2 were evaluated for in vivo cytotoxic potency in the HepG2-luc model. In these studies, mice were injected intravenously with one of the following: (1) 5×10 6 Non-transduced donor-matched (mock) primary human T cells, (2) 5×10 6 Primary human T cells expressing construct 1, (3) 2×10 6 Primary human T cells expressing construct 1, (4) 5×10 6 primary human T cells expressing construct 2, or (5) 2 × 10 6 Primary human T cells expressing construct 2 (n=10 mice / group) were generated. To simulate tumor recurrence, mice in all treatment groups underwent tumor re-challenge studies in which HepG2-luc cells were implanted subcutaneously in the left flank at approximately day 70 after T cell administration.

[0265] In all four treatment groups (i.e., high-dose construct 1, low-dose construct 1, high-dose construct 2, low-dose construct 2), tumors began to regress around day 10 after T cell injection; complete tumor regression was achieved around day 20 ( Figure 2 ). No significant differences were 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 "GPC3-caTCR+GPC3-CD30-CSR" T cells.

[0266] Interestingly, when animals underwent tumor re-challenge, an enhancing effect of c-Jun was observed. T cells expressing construct 2 had significantly better expression than T cells expressing construct 1, indicating that c-Jun enhanced the tumor killing potential of "GPC3-caTCR+GPC3-CD30-CSR" T cells in tumor re-challenge studies ( Figure 3 ). Additionally, in one study, mice injected with higher doses of T cells expressing construct 2 had higher peripheral blood T cell counts ( Figure 4A and Figure 4B ). Example 3: In vivo efficacy of anti-GPC3+anti-GPC3 CSR T cells overexpressing c-Jun in the Hep3B mouse model

[0267] This example demonstrates that T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR and overexpressing c-Jun increase T cell expansion in vivo compared to T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR without c-Jun.

[0268] The in vivo cytotoxic potency of primary human T cells expressing construct 1 or construct 2 was evaluated in the Hep3B model. Mice were injected intravenously with one of the following: (1) 2.5 × 10 6 Non-transduced donor-matched (mock) primary human T cells, (2) 2.5×10 6 Primary human T cells expressing construct 1, (3) 1.5×10 6 Primary human T cells expressing construct 1, (4) 2.5×10 6 primary human T cells expressing construct 2, or (5) 1.5 × 10 6 Primary human T cells expressing construct 2 (n = 10 mice / group). Figure 5 As shown in Figure 2, after initial regression, tumor regrowth was observed in all groups. T cells expressing construct 2 suppressed the size of recurrent tumors to a greater extent than T cells expressing construct 1. At the higher dose of 2.5 M, the difference between the tumor inhibitory effects of constructs 1 and 2 as measured by tumor volume was not discernible. In addition, mice in the construct 2 groups at both doses had higher numbers of peripheral blood T cells, indicating that c-Jun positively affects anti-GPC3 caTCR in vivo + Anti-GPC3 CSR + T cell expansion ( Figure 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 engineered to express various anti-GPC3 caTCRs comprising different sequences in addition to a human c-Jun polypeptide (e.g., GPC3-caTCR) and various anti-GPC3 CSRs comprising different sequences and a costimulatory domain derived from human CD30 (i.e., GPC3-CD30-CSR).

[0270] Primary T cells isolated from healthy human donors were transduced with one of the expression construct groups described in Table 4 below. Table 4. Exemplary expression constructs comprising different combinations of anti-GPC3 moieties

[0271] T cells expressing each set of expression constructs were co-cultured with HepG2 cells and analyzed for cytotoxicity and degree of depletion as described in Example 1 above.

[0272] The construct c-Jun+GPC3-3 caTCR+GPC3-2-CD30 CSR (Table 4) exhibited the highest long-term target cell killing capacity and the lowest degree of depletion compared to the other constructs described in Table 4. Example 5: Characterization of T cells expressing anti-GPC3 caTCR and different anti-GPC3 CSRs

[0273] This example features human T cells engineered to express an anti-GPC3 caTCR (e.g., GPC3-caTCR) in addition to a human c-Jun polypeptide and various anti-GPC3 CSRs (e.g., GPC3-CSRs) comprising different costimulatory fragments (e.g., costimulatory fragments comprising a costimulatory domain and a transmembrane domain).

[0274] Primary T cells isolated from healthy human donors were transduced with one of the expression constructs described in Table 5 below. Each exemplary GPC3-caTCR encodes the V H / V L sequence (see sequence listing), and each exemplary GPC3-CSR encodes the V H / V L Sequence (see Sequence Listing). Table 5. Exemplary expression constructs containing anti-GPC3-CSRs containing different co-stimulatory fragments

[0275] Comparative analysis of T cells expressing the constructs described in Table 5 (eg, T cells expressing the same anti-GPC3-caTCR (eg, GPC3-3 caTCR) and c-Jun, but with different anti-GPC3-CSRs comprising different co-stimulatory fragments) was performed and described in Examples 5A-5G.

[0276] Overall, Examples 5A-5G show that c-Jun+GPC3-3caTCR+GPC3-2-CD30 TM-CD30 IC-CSR and c-Jun+GPC3-3caTCR+GPC3-2-CD28 TM-CD30 IC-CSR performed equally well and better than other anti-GPC3-CSRs. Example 5A: Short-term in vitro cancer cell killing assay

[0277] T cells expressing each of the constructs described in Table 5 were co-cultured with HepG2 cells and cytotoxicity was determined by Cytox as described in Example 1. Tumor cytotoxicity was analyzed using a non-radioactive cytotoxicity assay (Promega) that quantitatively measures lactate dehydrogenase (LDH) released into the culture supernatant.

[0278] T cells expressing 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 had higher killing efficacy than other constructs without CSR or CSR containing other co-stimulatory fragments as summarized in Table 5.

[0279] The short-term killing ability of various T cells expressing the constructs of Table 5 is also determined by measuring the amount / content of cytokines released from T cells after engaging with HepG2 cells. The content of cytokine release in the supernatant after 16 hours of co-cultivation is quantitatively measured using the BioRad Bio-Plex test kit or by ELISA using Luminex Magpix technology. T cells with higher cytotoxicity secrete high levels of cytokines associated with T cell activity, such as TNFα, GM-CSF, IFNγ and IL-2.

[0280] T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR comprising at least the CD30 IC domain had higher killing efficacy than those T cells without any CSR, and higher than or about the same as those T cells containing CSRs that did not comprise the CD30 IC domain but had a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain) (see Table 5). Example 5B: Proliferation Potential and Persistence Analysis

[0281] The proliferation and persistence of genetically modified T cells are crucial for the success of adoptive T cell transfer therapy when treating cancer. To analyze the effects of specific CSRs on T cell proliferation and persistence, T cells expressing each of the constructs in Table 5 were labeled with the intracellular dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and when T cells divide under tumor cell stimulation, the dye is observed to be diluted. The persistence of T cells is measured by counting the number of CFSE-positive cells remaining on a given day.

[0282] T cells expressing each of the constructs shown in Table 5 were serum starved overnight and labeled with CFSE using CellTrace CFSE (Thermo Fisher). 50,000 to 100,000 T cells were cultured at a 2:1 effector cell to target cell ratio (E:T ratio), and when the T cells split on the specified day, serial dilutions of CFSE dye were observed using flow cytometry. The total number of T cells was counted using FACS.

[0283] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain proliferate to a greater extent than corresponding CAR T cells without any CSRs, and proliferate to a greater extent than or about the same as corresponding CAR T cells expressing a CSR that does not comprise a CD30 IC domain but has a different costimulatory molecule IC domain, such as a CD28 or 4-1BB IC domain. Example 5C: In vitro T cell and tumor cell counts after multiple weeks of conjugation

[0284] FACS-based analysis for target cell enumeration was 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 exhibited comparable survival rates after 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 persisted in multiple engagements with tumor cells for longer periods of time and killed more tumor cells than corresponding CAR T cells without a CSR, and were about the same as (if not better than) CAR T cells containing a CSR without a CD30 IC domain but with an IC domain of a different co-stimulatory molecule, such as a CD28 or 4-1BB IC domain.

[0286] FACS-based analysis for counting T cells and target cells was used to compare the long-term survival and target cell killing potential of T cells expressing the constructs described in Table 5. T cells were co-cultured and re-challenged with target cells as described in Example 1. At various days after each target cell engagement, the number of remaining target cells and total T cells was quantified by FACS.

[0287] T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR comprising at least a CD30 IC domain persist / survive through multiple engagements of tumor target cells for longer periods of time and kill more tumor cells than corresponding T cells without any CSRs, and survive better and / or kill more tumor cells than or about the same as corresponding T cells expressing a CSR without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., a CD28 or 4-1BB IC domain). Example 5D: In vitro T cell depletion

[0288] To examine the levels of exhaustion markers expressed on T cells transduced with the constructs described in Table 5, caTCR + CD8 + T cells and caTCR + CD4 + Exhaustion markers on T cells: TIGIT, PD-1, LAG-3 and TIM-3 The expression levels of were analyzed by flow cytometry at 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 the CD30 IC domain have reduced long-term exhaustion markers compared to corresponding T cells without any CSR or T cells expressing a CSR without the CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5E: Cytokine Release in Vivo

[0290] As described in Example 2 and Example 3, respectively, primary human T cells expressing each of the constructs in Table 5 were evaluated for in vivo cytotoxicity in the HepG2-luc and Hep3B mouse models. Briefly, to determine the level of cytokine release in vivo, key cytokines, including those associated with clinical cytokine release syndrome, were analyzed 16, 24, 48, and 72 hours after administration of T cells. Cytokine levels were quantified by Luminex Magpix technology using the BioRad Bio-Plex kit.

[0291] T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR comprising at least the CD30 IC domain secrete higher levels of cytokines associated with T cell activity, such as TNFα, GM-CSF, IFNγ, and IL-2, than corresponding T cells without any CSR or T cells expressing a CSR without the CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5F: Differentiation of T cell subsets over time (CCR7 / CD45RA) and quantification of memory T cells

[0292] In two independent flow cytometric analyses, proliferation and survival of T cells expressing the expression constructs described in Table 5 were measured before and after target cell engagement.

[0293] FACS analysis of T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR comprising at least a CD30 IC domain showed higher expression of the T cell differentiation markers CCR7 and CD45RA and increased percentages of memory T cells and naive T cells compared to T cells without any CSR or T cells expressing a CSR without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain).

[0294] T cells expressing c-Jun, anti-GPC3 caTCR and anti-GPC3-CD30-CSR develop into and maintain a high amount of memory T cell populations after target stimulation, including central memory T cells and effector memory T cells. To determine the effects of different CSRs on the ability of T cells to develop and maintain memory T cells, the cell surface expression of memory T cell markers CCR7 and CD45RA is measured. As known in the art, T cells with high CCR7 expression and low CD45RA expression are regarded as central memory T cells, T cells with low CCR7 and low CD45RA expression are regarded as effector memory T cells, T cells with low CCR7 and high CD45RA expression are regarded as effector T cells, and T cells with high CCR7 and high CD45RA are naive T cells, i.e., T cells of the initial type before targeting / antigen attack / recognition (Mahnke et al., Eur J Immunol. 43 (11): 2797-809, 2013). In response to antigen encounter, naive T cells proliferate and differentiate into effector cells, most of which perform the work of destroying the target and then die, while a small pool of T cells eventually develops into long-term memory T cells that can store T cell immunity against specific targets. Among memory T cells, central memory T cells are found to have a longer lifespan than effector memory T cells and are able to generate 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 of possible successful T cell therapy.

[0295] T cells expressing c-Jun and anti-GPC3 caTCR but without CSR were incubated with HepG2 cells at an E:T ratio of 2:1 (e.g., 100,000 receptors per well of a 96-well plate). + T cells and 50,000 HepG2 cells per well) were incubated for 7 days. The cells were then challenged with 50,000-100,000 HepG2 cells per well every 7 days. T cells in other groups expressing different CSRs were challenged with target cells at an E:T ratio of 1:2 (e.g., 25,000 receptors per well). + T cells and 50,000 HepG2 cells) were incubated together for 7 days. The cells were then re-challenged every 7 days with 50,000-100,000 HepG2 cells per well.

[0296] In some experiments, the T cell and HepG2 cell mixture was diluted 1:6 before the fourth and fifth HepG2 cell junctions to avoid T cell overcrowding due to significant T cell expansion, so that only one-sixth of the previous remaining cells were re-challenged by 50,000-100,000 HepG2 cells.

[0297] On selected days after each HepG2 cell incorporation, the entire cell mixture from the wells of each sample was stained with antibodies against CCR7 and CD45RA and analyzed by flow cytometry. + The number of T cells was counted and the cells were grouped into various T cell types based on the expression of CCR7 and CD45RA: central memory T cells (CD45RA - CCR7 + ), effector memory T cells (CD45RA - CCR7 - ), effector T cells (CD45RA+CCR7 - ) and primary T cells (CD45RA + CCR7 + ). Calculate receptor + The percentage of each type of T cell among the total number of T cells. In some experiments, cells were also stained with antibodies against CD8 or CD4 to determine the composition of CD8 (cytotoxic T cells) and CD4 (helper T cells) T cell types.

[0298] The proliferation and survival of T cells expressing c-Jun, anti-GPC3 caTCR and anti-GPC3 CSR were measured before and after target cell engagement. T cells expressing c-Jun, anti-GPC3 caTCR and anti-GPC3 CSR comprising at least a CD30 IC domain were able to develop and maintain high numbers and percentages of central memory T cells after engagement with HepG2 cells, higher than T cells without any CSR or T cells expressing CSRs without a CD30 IC domain but with a different costimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5G: In vivo efficacy of T cells in HepG2 or Hep3B mouse models

[0299] In NSG mice, approximately 10 7 HepG2 tumor cells or Hep3B and allowed to form 150mm 3 Solid tumor mass. 5×10 6 T cells were injected intravenously into tumor-bearing mice. Three weeks after T cell administration, mice were sacrificed and tumors were removed, fixed and sliced ​​onto slides. Tumor sections were stained with CD3 antibody to observe T cells present in solid tumors. + Quantification of cell number can be used to assess the tumor infiltration capacity of T cells (T cells / mm 2 ) for scoring.

[0300] T cells expressing c-Jun, anti-GPC3 caTCR and anti-GPC3 CSR comprising at least CD30 IC domain have higher in vivo tumor infiltration / penetration / content (i.e., higher number of T cells / mm) than T cells without any CSR or T cells expressing CSR without CD30 domain but with different costimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). 2 ). Sequence Listing

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 moiety 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 promotes the recruitment of at least one TCR-associated signaling molecule; b) a chimeric stimulatory receptor (CSR) comprising: i) a ligand binding module capable of binding to or interacting with GPC3; ii) a transmembrane module; and iii) a costimulatory immune cell signaling module capable of providing a costimulatory signal to an 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) Human c-Jun polypeptide.

2. The expression construct of claim 1, wherein the c-Jun is wild-type human c-Jun.

3. The expression construct of claim 2, wherein the wild-type human c-Jun has at least about 90% identity to the amino acid sequence of SEQ ID NO:

1.

4. The expression construct of claim 1, wherein the c-Jun is a mutant human c-Jun.

5. The expression construct of claim 4, wherein the mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain.

6. The expression construct of claim 4 or 5, wherein the mutant human c-Jun comprises: (i) S63A and S73A substitutions 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 compared to SEQ ID NO:

1.

7. The expression construct of any one of claims 1 to 6, wherein the GPC3 is cell surface-bound GPC3.

8. The expression construct of any one of claims 1 to 7, wherein the TCR M is derived from a human γ / δ TCR.

9. The expression construct of any one of claims 1 to 8, wherein the caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first stabilization domain and the second stabilization domain have a binding affinity to each other that stabilizes the caTCR.

10. The expression construct of claim 9, wherein the stabilizing module is selected from the group consisting of: a TCR constant domain, a C H 1-C L Module, C H 2-C H 2 modules, C H 3-C H 3 modules and C H 4-C H 4 modules.

11. The expression construct of claim 10, wherein the stabilizing moiety is derived from a human protein.

12. The expression construct of claim 10 or 11, wherein the C H 1-C L The C included in the module L Derived from a kappa antibody light chain or a lambda antibody light chain.

13. The expression construct of any one of claims 1 to 12, wherein the antigen binding moiety of the caTCR is Fab, Fab', (Fab')2, Fv or single chain Fv (scFv).

14. The expression construct of any one of claims 1 to 13, wherein the antigen binding moiety of the caTCR comprises: (i) a heavy chain variable domain (V) comprising a heavy chain complementary 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, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8 H ); and a light chain variable domain (VLD) comprising a light chain complementary 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, a LC-CDR2 comprising the amino acid sequence of GDN or a variant thereof comprising up to about 3 amino acid substitutions, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 3 amino acid substitutions. L ); (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L ;or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L .

15. The expression construct of claim 14, wherein the antigen binding moiety of the caTCR comprises: (i) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a LC-CDR2 comprising the amino acid sequence of GDN, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 L ; (ii) a V comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 H and a V 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 L ;or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 L .

16. The expression construct of any one of claims 1 to 15, wherein the antigen binding moiety of the caTCR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 24 H , and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:25 L ; (ii) a V comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 26 H , and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:27 L ;or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 28 H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:29 L .

17. The expression construct of claim 16, wherein the antigen binding moiety of the caTCR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ; (ii) V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;or (iii) V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L .

18. The expression construct of any one of claims 1 to 17, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising a first TCRD and a second polypeptide chain comprising a second TCRD.

19. The expression construct of any one of claims 1 to 18, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a V fused to a TCRD derived from a TCRδ chain. H , the second polypeptide chain comprises a V fused to a TCRD derived from a TCRγ chain L .

20. The expression construct of any one of claims 1 to 18, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a V fused to a TCRD derived from a TCRγ chain. H , the second polypeptide chain comprises a V fused to a TCRD derived from a TCRδ chain L .

21. The expression construct of any one of claims 1 to 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 having at least about 90% sequence identity with SEQ ID NO:30, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

31.

22. The expression construct of any one of claims 1 to 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 having 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 having at least about 90% sequence identity to SEQ ID NO:

5.

23. The expression construct of any one of claims 1 to 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 having at least about 90% sequence identity with SEQ ID NO:63, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

64.

24. The expression construct of any one of claims 1 to 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 having at least about 90% sequence identity with SEQ ID NO:65, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

66.

25. The expression construct of any one of claims 1 to 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 having at least about 90% sequence identity with SEQ ID NO:67, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:68 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

68.

26. The expression construct of any one of claims 1 to 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 having at least about 90% sequence identity with SEQ ID NO:69, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:70 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

70.

27. The expression construct of any one of claims 1 to 26, wherein the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

28. The expression construct of any one of claims 1 to 27, wherein the transmembrane module of the CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.

29. The expression construct of any one of claims 1 to 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 of claim 29, wherein the co-stimulatory receptor is selected from the group consisting of CD30, CD28, 4-IBB, OX40, ICOS, CD27 and CD40.

31. The expression construct of any one of claims 1 to 30, wherein the co-stimulatory immune cell signaling module of the CSR is derived from human CD30.

32. The expression construct 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 of any one of claims 1 to 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 of any one of claims 1 to 33, wherein the ligand binding module of the CSR comprises: (i) a V sequence comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8; H and a V 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 L ; (ii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L ;or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising up to about 3 amino acid substitutions, a 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 a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to about 3 amino acid substitutions. H and a V 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 L .

35. The expression construct of claim 34, wherein the ligand binding module of the CSR comprises: (i) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a LC-CDR2 comprising the amino acid sequence of GDN, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 L ; (ii) a V comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 H and a V 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 L ;or (iii) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 H and a V comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a LC-CDR2 comprising the amino acid sequence of DDS, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 L .

36. The expression construct of any one of claims 1 to 35, wherein the ligand binding module of the CSR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 24 H , and V comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:25 L ; (ii) a V comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 26 H , and V comprising the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:27 L ;or (iii) a V comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 28 H , and V comprising the amino acid sequence of SEQ ID NO:29 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:29 L .

37. The expression construct of claim 36, wherein the ligand binding module of the CSR comprises: (i) a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ; (ii) V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;or (iii) V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L .

38. The expression construct of any one of claims 1 to 37, wherein the ligand binding module of the CSR comprises: (i) SEQ ID NO:32 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:32; (ii) SEQ ID NO:33 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:33; or (iii) SEQ ID NO:34 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:

34.

39. The expression construct of any one of claims 1 to 38, wherein: (i) the antigen binding module of the caTCR comprises: (a) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 H ;as well as (b) a V comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR2 comprising the amino acid sequence of DDS, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23 L ;as well as (ii) the ligand binding module of the CSR comprises: (a) a V comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14 H ;as well as (b) a V comprising LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, LC-CDR2 comprising the amino acid sequence of YDS, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17 L .

40. The expression construct of any one of claims 1 to 39, wherein the antigen binding moiety of the caTCR comprises a Fab that binds GPC3, wherein the ligand binding moiety of the CSR comprises a scFv that binds GPC3, and wherein the transmembrane moiety and the co-stimulatory immune cell signaling moiety of the CSR are both derived from CD30.

41. The expression construct of claim 40, wherein the CSR comprises a fragment of CD30, the fragment comprising the amino acid sequence of SEQ ID NO: 43, and wherein: (1)(i) The Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; (2) (i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; (3) (i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ; (4)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ; (5)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ; (6)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ; (7)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ; (8)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;or (9)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L .

42. The expression construct of claim 40 or 41, wherein the CSR comprises a fragment of CD30, the fragment comprising the amino acid sequence of SEQ ID NO: 43, and wherein: (1)(i) The Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; (2) (i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32; (3) (i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 28 H and V comprising the amino acid sequence of SEQ ID NO: 29 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34; (4)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32; (5)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; (6)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 26 H and V comprising the amino acid sequence of SEQ ID NO: 27 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34; (7)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32; (8)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; or (9)(i) the Fab comprises a V comprising the amino acid sequence of SEQ ID NO: 24 H and V comprising the amino acid sequence of SEQ ID NO:25 L ;as well as (ii) the scFv comprises the amino acid sequence of SEQ ID NO:

34.

43. The expression construct of any one of claims 1 to 42, wherein the construct is a viral vector.

44. The expression construct of claim 43, wherein the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex viral vector, and an Epstein-Barr viral vector.

45. The expression construct of any one of claims 1 to 42, wherein the expression construct comprises a polycistronic expression cassette for expressing the caTCR, the CSR and the c-Jun.

46. ​​A multicistronic expression construct comprising an expression cassette for expressing: a) a chimeric antibody T cell receptor (TCR) construct (caTCR) comprising: i) an antigen binding moiety that specifically binds to glypican 3 (GPC3); and ii) TCR module (TCRM) derived from human γ / δ TCR; b) a chimeric stimulatory receptor (CSR) comprising: i) a ligand binding module capable of binding to or interacting with GPC3; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module derived from the intracellular domain of human CD30; and c) Human c-Jun polypeptide.

47. The expression construct of claim 46, wherein the expression cassette comprises the coding sequence of SEQ ID NO: 1 and: (i) the coding sequences of SEQ ID NOs: 28 and 29 and the coding sequence of SEQ ID NO: 33; (ii) the coding sequences of SEQ ID NOs: 28 and 29 and the coding sequence of SEQ ID NO: 32; (iii) the coding sequences of SEQ ID NOs: 28 and 29 and the coding sequence of SEQ ID NO: 34; (iv) the coding sequences of SEQ ID NOs: 26 and 27 and the coding sequence of SEQ ID NO: 32; (v) the coding sequences of SEQ ID NOs: 26 and 27 and the coding sequence of SEQ ID NO: 33; (vi) the coding sequences of SEQ ID NOs: 26 and 27 and the coding sequence of SEQ ID NO: 34; (vii) the coding sequences of SEQ ID NOs: 24 and 25 and the coding sequence of SEQ ID NO: 32; (viii) the coding sequence of SEQ ID NOs: 24 and 25 and the coding sequence of SEQ ID NO: 33; or (ix) the coding sequences of SEQ ID NOs: 24 and 25 and the coding sequence of SEQ ID NO:

34.

48. The expression construct of claim 47, wherein the coding sequence is separated in frame by a 2A coding sequence or by an internal ribosome entry site (IRES).

49. The expression construct of any one of claims 46 to 48, wherein the expression cassette comprises a constitutive or inducible promoter.

50. The expression construct of claim 49, wherein the promoter is the EF-1 alpha promoter.

51. The expression construct of any one of claims 46 to 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 a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia vector, a herpes simplex viral vector, and an Epstein-Barr viral vector.

53. A recombinant virus comprising the multicistronic expression construct of any one of claims 50 to 52.

54. The recombinant virus of claim 53, wherein the polycistronic expression construct is a lentiviral vector.

55. A method for engineering immune cells, comprising: (a) providing a starting cell population, (b) introducing the expression construct of any one of claims 1 to 52 or the recombinant virus of claim 53 or 54 into the starting cell population, (c) optionally selecting cells expressing said caTCR, said CSR and said c-Jun, and (d) obtaining 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 cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.

58. The method of any one of claims 55 to 57, wherein the starting cell population comprises immune cells.

59. The method of any one of claims 55 to 58, wherein the starting cell population comprises autologous or allogeneic T cells.

60. The method of any one of claims 55 to 57, wherein the starting cell population comprises multipotent or pluripotent 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 of any one of claims 1 to 52 or the recombinant virus of claim 53 or 54.

62. The human cell population of claim 61, wherein the human cells are immune cells.

63. An immune cell population obtained by the method of any one of claims 55 to 60.

64. The immune cell population of claim 63, wherein the immune cells are human cells.

65. The immune cell of any one of claims 62 to 64, wherein the immune cell is a T cell.

66. The immune cell of claim 65, wherein the T cell is a CD8 + T cells.

67. The immune cell of any one of claims 62 to 66, wherein the immune cell expresses a lower amount of an exhaustion marker than a corresponding cell that does not overexpress c-Jun.

68. The immune cell 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 of any one of claims 1 to 52, the recombinant virus of claim 53 or 54, or the cell of any one of claims 61 to 68 and a pharmaceutically acceptable carrier.

70. A method of killing a target cell, comprising contacting the target cell with the pharmaceutical composition of claim 69 under conditions that allow killing of the target cell by an immune cell, wherein the target cell expresses GPC3.

71. The method of claim 70, wherein the immune cell expresses a lower amount of the exhaustion marker when contacted with the target cell compared to a corresponding immune cell that does not contain the exogenous nucleic acid molecule that causes overexpression of c-Jun.

72. The method of claim 70 or 71, wherein the immune cell is a T cell.

73. The method of claim 72, wherein the T cells are CD8 + T cells.

74. The method of claim 73, wherein the target cell is a cancer cell.

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 cell expresses a lower amount of an exhaustion marker than a corresponding immune cell engineered to express a CSR in which the co-stimulatory immune cell signaling module is derived from human CD28.

76. The method of any one of claims 70 to 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 to the individual the pharmaceutical composition of claim 69.

78. Use of the expression construct of any one of claims 1 to 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 of any one of claims 1 to 52, the recombinant virus of claim 53 or 54, or the pharmaceutical composition of claim 69 for treating a subject in need thereof.

80. The method of claim 77 or the use of claim 78 or 79, wherein the individual suffers from 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, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, Wilms' tumor, testicular non-seminomatous germ cell tumor, gastric cancer, and liposarcoma.

83. A method for reducing exhaustion of an engineered immune cell, comprising introducing into 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 of any one of claims 1 to 52.

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