TW202413646A - Immune cell therapy - Google Patents

Abstract

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

Description

Immunocell Therapy

In the past two decades, fundamental advances in immunology and tumor biology and the identification of a large number of tumor antigens have led to remarkable 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 into patients, has induced some remarkable anti-tumor responses (Blattman et al., Science . (2004) 305(5681):200-5). For example, administration of ex vivo expanded naturally occurring tumor infiltrating lymphocytes (TILs) modulated objective response rates in the 50-70% range in melanoma patients, including patients with large, aggressive tumors at multiple sites involving the liver, lungs, soft tissue, and brain (Rosenberg et al., Nat Rev Cancer. (2008) 8(4):299-308; Dudley et al., J Clin Oncol. (2005) 23(10):2346-57).

The major limitation to 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 the patient's normal autologous T cells through T cell engineering. The transfer of these cells into lymphocyte-depleted patients 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 that are processed and delivered to the cell surface via MHC presentation; these antigens can be recognized by antigen-specific cytotoxic T cells even at low densities (Kunert, supra).

Several attempts have been made to engineer TCR molecules with antibody specificity and T cell receptor effector function. In some of these methods, the variable and constant domains of a TCR (e.g., an αβ TCR or a γδ TCR) are replaced with the variable and constant domains of an antibody against a tumor antigen, thereby generating a chimeric antibody TCR, referred to as an "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 these methods, a chimeric stimulatory receptor (CSR) is used in combination with a caTCR to enhance the tumor killing efficacy of engineered T cells. Like chimeric antigen receptors (CARs), CSRs have an extracellular domain that binds a target ligand (e.g., a tumor antigen) and an intracellular synergistic stimulatory domain, but unlike CARs, CSRs do not have an intracellular primary immune cell signaling domain (which is typically the intracellular domain of the CD3 ζ chain) or a functional primary immune cell signaling domain. CSRs and caTCRs can bind different antigenic determinants 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.

One challenge facing T cell therapy is the lack of persistence of T cells in vivo due to a phenomenon called T cell exhaustion. See, e.g., Fraietta et al., Nat Med . (2018) 24(5):563-71; Long et al., Nat Med . (2015) 21(6):581-90; and Eyquem et al., Nature (2017) 543(7643):113-7. T cell exhaustion is characterized by significant changes in metabolic function, transcriptional programming, loss of effector function (e.g., decreased interleukin secretion and cytotoxicity), expression of multiple surface inhibitory receptors, and apoptosis. T cell exhaustion is due to constant antigen exposure, resulting in persistent TCR signaling, or tonic antigen-independent signaling via engineered antigen receptors on T cells (see, e.g., Long, supra). Preventing or reversing T cell exhaustion has been sought as a means of enhancing T cell effectiveness, e.g., in patients with cancer or chronic infection and in T cell therapy. See, e.g., WO 2019/118902, the disclosure of which is incorporated herein by reference in its entirety.

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

Therefore, there remains a need for improved T cell therapies in which engineered T cells have higher and sustained tumor-killing potency, and in particular, improved T cell therapies in which T cells specifically target cancers that overexpress GPC3.

The present invention provides compositions and methods for improving immunocytotherapy. In some aspects, one or more expression constructs are provided herein, comprising one or more expression cassettes for expressing: a) a chimeric antibody T cell receptor (TCR) construct (caTCR) comprising: i) an antigen binding module that specifically binds to glypican 3 (GPC3); and ii) a) 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-related signaling molecule; b) a chimeric stimulatory receptor (CSR) comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a synergistic immune cell signaling module capable of providing a synergistic stimulatory signal to the immune cell, wherein the ligand binding module and the synergistic 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, 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.

In some embodiments, GPC3 is cell surface-bound GPC3.

In some embodiments, the TCR 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 and second stabilizing domains 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- _CL module, a C _H 2- _CH 2 module, a C _H 3- _CH 3 module, and a C _H 4- _CH 4 module. In some embodiments, the stabilizing module is derived from a human protein. In some embodiments, the _CL contained in the C _H 1- _CL module is derived from a κ antibody light chain or a λ antibody light chain. In some embodiments, the antigen binding module of the caTCR is Fab, Fab', (Fab') ₂ , Fv or single-chain Fv (scFv).

In some embodiments, the antigen binding module of the caTCR comprises: (i) a heavy chain variable domain (VH) 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; and 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 HC-CDR3 comprising the amino acid sequence of SEQ ID NO: (i) a light chain variable domain ( _VL ) comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising at most about 3 amino acid substitutions, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising at most about 3 amino acid substitutions, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising at most about 3 amino acid substitutions; and a _{VH comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising at most about 3 amino acid substitutions, an LC-CDR2 comprising an amino acid sequence of YDS or a variant thereof comprising at most about 3 amino acid substitutions, and an HC} -CDR3 comprising an amino acid sequence of SEQ ID NO: 16 or a variant thereof comprising at most about 3 amino acid substitutions. or (iii) _{a VL} comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising at most about 3 amino acid substitutions; and a _VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising at most about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of DDS or a variant thereof comprising at most about 3 amino acid substitutions, and a VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising at most about 3 amino acid substitutions, 23 amino acid sequence or a _variant thereof comprising up to about 3 amino acid substitutions. In some embodiments, the antigen binding module of the caTCR comprises: (i) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 8; and a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising an amino acid sequence of GDN, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11; (ii) a _{VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14; and a VL} comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising an amino acid sequence of _GDN , and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11. or (iii) a _{VH 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; and a VL 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.

In some embodiments, the antigen-binding module of the caTCR comprises: (i) a _VH comprising an 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, and a _VL comprising an 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; (ii) a _{VH comprising an 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, and a VL} comprising an 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; or (iii) a _VH comprising an 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, and a _VL comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: In some embodiments, the antigen binding module of the caTCR comprises: (i) a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of _SEQ ID NO: 27; or (iii) a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, 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, caTCR is a heterodimer comprising a first polypeptide chain comprising a _VH fused to a TCRD derived from a TCR delta chain and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR gamma chain. In some embodiments, caTCR is a heterodimer comprising a first polypeptide chain comprising a _VH fused to a TCRD derived from a TCR gamma chain and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR delta chain. In some embodiments, the caTCR is a heterodimer comprising: a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 30; and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising: a first polypeptide chain comprising an 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 an 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. In some embodiments, the caTCR is a heterodimer comprising: a first polypeptide chain comprising an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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.

In some embodiments, the transmembrane module of 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 CSR comprises a transmembrane domain derived from the transmembrane domain of CD30.

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

In some embodiments, the ligand binding module of CSR comprises: (i) a heavy chain variable domain (VH) comprising a heavy chain complementary determining region (HC-CDR) 1 (HC-CDR1) comprising an amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 3 amino acid substitutions, a HC-CDR2 comprising an 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 an amino acid sequence of SEQ ID NO: 8; and a light chain complementary determining region (LC- _CDR ) 1 (LC-CDR1) comprising an amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of GDN or a variant thereof comprising up to about 3 amino acid substitutions, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 10. (i) a light chain variable domain ( _VL ) comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising at most about 3 amino acid substitutions, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising at most about 3 amino acid substitutions, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising at most about 3 amino acid substitutions; and a _{VH comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising at most about 3 amino acid substitutions, an LC-CDR2 comprising an amino acid sequence of YDS or a variant thereof comprising at most about 3 amino acid substitutions, and an HC} -CDR3 comprising an amino acid sequence of SEQ ID NO: 16 or a variant thereof comprising at most about 3 amino acid substitutions. or (iii) _{a VL} comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising at most about 3 amino acid substitutions; and a _VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising at most about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of DDS or a variant thereof comprising at most about 3 amino acid substitutions, and a VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising at most about 3 amino acid substitutions, 23 amino acid sequence or a _variant thereof comprising up to about 3 amino acid substitutions. In some embodiments, the ligand binding module of CSR comprises: (i) a VH comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 6, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 7, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 8; and a _VL comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, a LC-CDR2 comprising an amino acid sequence of GDN, and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11; (ii) a _VH 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; and a _{VL comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, a LC-CDR2 comprising an amino acid sequence of GDN} , and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11. or (iii) a _{VH 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; and a VL 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.

In some embodiments, the ligand binding module of the CSR comprises: (i) a _VH comprising an 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, and a _VL comprising an 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; (ii) a _VH comprising an 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, and a VL comprising an 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; or (iii) a _VH comprising an 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, and a _VL comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: In some embodiments, the ligand binding module of the CSR comprises: (i) a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a _VH comprising the amino acid sequence of SEQ ID NO: 26 and _{a VL comprising} the amino acid sequence of SEQ ID NO: 27; or (iii) a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29.

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 with SEQ ID NO: 32; (ii) SEQ ID NO: 33 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 33; or (iii) SEQ ID NO: 34 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 34.

In some embodiments, (i) the antigen-binding module of the caTCR comprises: (a) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20; and (b) a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising an amino acid sequence of DDS, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23; and (ii) the ligand-binding module of the CSR comprises: (a) a _{VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14; and (b) a VL} comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising an amino acid sequence of _DDS , and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23. The V _L of 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.

In some embodiments, the antigen binding module of the caTCR comprises a Fab that binds GPC3, wherein the ligand binding module of the CSR comprises a scFv that binds GPC3, and wherein the transmembrane module of the CSR and the synergistic immune cell signaling module are both derived from CD30 (e.g., human CD30).

In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34.

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 viral vector, and an Epstein-Barr viral vector.

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

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 human γ/δ TCR; b) a chimeric stimulatory receptor (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 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 NOs: 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 NOs: 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 sequence of SEQ ID NOs: 24 and 25, and the coding sequence of SEQ ID NO: 34.

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 described in any of the foregoing embodiments. In some embodiments, the polycistronic expression construct is a lentiviral vector.

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 described in any of the foregoing technical solutions or a recombinant virus as described in any of the foregoing technical solutions into the starting cell population; (c) selecting cells expressing caTCR, CSR, and c-Jun as appropriate; 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, the T cells are selected from a 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 pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells.

In some aspects, a human cell population is provided herein, comprising an expression construct of any one of the foregoing embodiments or a recombinant virus of any one of the foregoing embodiments. In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell population is obtained by a method as described in any one of the foregoing embodiments. In some embodiments, the immune cell is a human cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8 ⁺ T cell. In some embodiments, the immune cell expresses a lesser amount of exhaustion markers than a corresponding cell that does not overexpress c-Jun. In some embodiments, the exhaustion marker is selected from a group consisting of CD39, PD-1, TIM-3, and LAG-3.

In some aspects, provided herein is a pharmaceutical composition comprising an expression construct as described in any of the foregoing embodiments, a recombinant virus as described in any of the foregoing embodiments, or a cell as described in any of the foregoing embodiments and a pharmaceutically acceptable carrier.

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 less depletion markers 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 cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the synergistic stimulatory immune cell signaling module in the CSR is derived from human CD30, and the immune cells express less exhaustion markers than corresponding immune cells engineered to express a CSR in which the synergistic stimulatory immune cell signaling module is derived from human CD28. In some embodiments, the exhaustion marker is selected from the group consisting of CD39, PD-1, TIM-3, and LAG-3.

In some aspects, provided herein is a method of treating an individual in need thereof, comprising administering to the individual a pharmaceutical composition as in any of the foregoing embodiments. In some aspects, provided herein is a 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 for the manufacture of a medicament for treating an individual in need thereof. In some aspects, provided herein is a 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 for treating an individual in need thereof. 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.

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 the expression construct of any of the preceding embodiments.

Cross-references to related applications

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.

The present application provides an engineered human cell (e.g., an immune cell, such as a T cell) comprising a construct 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 gastrointestinal cancer, such as gastric cancer or liver cancer. The anti-GPC3 caTCR is regulated by a naturally occurring mechanism that controls TCR activation, and 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.

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

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 overexpression of c-Jun significantly reduced the depletion of T cells engineered to express anti-GPC3 caTCR and CD30-based anti-GPC3 CSR. I. Definitions

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 complementary 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 flanking segments called framework regions (FRs), which are more highly conserved than the 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 major 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. Some of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, a bifunctional antibody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized bifunctional antibody (dsbifunctional antibody), a single-chain antibody molecule (scFv), an scFv dimer (a bivalent bifunctional antibody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camel-like single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to framework regions from one or more different human antibodies.

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 a target (which may be an antigenic determinant) 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 reacts with a binding affinity for one or more antigenic determinants of an antigen (e.g., a cell surface antigen or a peptide/MHC protein complex) that is at least about 10 times greater than its binding affinity for other targets.

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 is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition via complementation determining regions (CDRs), followed by a constant domain (C) that is anchored to the cell membrane by a linking 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. The complex interaction between these CDRs and antigenic peptides bound 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).

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

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

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, the 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, the 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.

As used herein, the term "isolated nucleic acid" means a nucleic acid of genomic, cDNA or synthetic origin, or a combination thereof, by which the "isolated nucleic acid" (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 naturally linked, or (3) does not occur as part of a larger sequence in nature.

As used herein, the term "CDR" or "complementary determining region" refers to non-contiguous antigen combining sites found within the variable regions of 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., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest"(1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol. , 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol ., 45: 3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol. , 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol. , 309:657-670 (2001), 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 the CDRs or variants thereof of an antibody or a grafted antibody is intended to be within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined in each of the above cited references are described below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol. , 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res. , 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res. , 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in the present invention and may be included in one or more technical solutions herein. The specific CDR sequences of the present invention are defined by the IMGT numbering system. Table 1. CDR definitions . Kabat ¹ Chothia ² MacCallum ³ IMGT ⁴ AHo ⁵ _VH CDR1 31-35 26-32 30-35 27-38 25-40 _VH CDR2 50-65 53-55 47-58 56-65 58-77 _VH CDR3 95-102 96-101 93-101 105-117 109-137 V _L CDR1 24-34 26-32 30-36 27-38 25-40 V _L CDR2 50-56 50-52 46-55 56-65 58-77 V _L CDR3 89-97 91-96 89-96 105-117 109-137 ¹ Residue numbering follows the nomenclature of Kabat et al. ² Residue numbering follows the nomenclature of Chothia et al ^{. 3} Residue numbering follows the nomenclature of MacCallum et al. ⁴ Residue numbering follows the nomenclature of Lefranc et al. ⁵ Residue numbering follows the nomenclature of Honegger and Plückthun

The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and/or the light chain is identical or homologous to the 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 the 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. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA , 81:6851-6855 (1984)).

The term "semisynthetic" with reference 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.

The term "fully synthetic" with respect to an antibody or antibody portion means that the antibody or antibody portion has a fixed naturally occurring _VH / _VL framework pairing, but rather than non-naturally occurring (i.e., synthetic) sequences of all six CDRs of both the heavy and light chains.

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

"Single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising _VH and _VL antibody domains linked in a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the _VH and _VL 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).

"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human antibodies. To a large extent, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the hypervariable regions (HVRs) of the recipient are replaced with residues in the hypervariable regions of a non-human species (donor antibodies), such as mice, rats, rabbits, or non-human primates that have the desired antibody specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region, as appropriate. For further details, see Jones et al. , Nature 321:522-525 (1986); Riechmann et al. , Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) .

"Homology" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When one position in the two sequences being compared is occupied by the same base or amino acid monomer subunit, for example, if one position in each of the two DNA molecules is occupied by adenine, then the molecules are "homologous" at that position. The "percent homology" or "percent sequence identity" between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions times 100, with any conservative substitutions considered part of the sequence identity. For example, if 6 out of 10 positions in two sequences are matched or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. In general, a comparison is made when two sequences are aligned for maximum homology. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes of this article, the sequence alignment 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).

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

The term "operably linked" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, thereby causing the latter to express. 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. Generally speaking, operably linked DNA sequences are contiguous and, if necessary, connect two protein coding regions in the same reading frame.

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 inducer or conditions that activate the promoter and/or relieve the repression of the promoter.

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.

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 a disease, reducing the scope of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or reducing the worsening of the disease, improving the disease state, causing the disease to be relieved (partially or completely), reducing the dosage of one or more other agents required to treat the disease, delaying the worsening of the disease, enhancing or improving the quality of life, increasing the amount of weight gain and/or prolonging survival. "Treatment" also encompasses the reduction of pathological consequences of a disease (such as tumor size in cancer). The methods of the present invention encompass any one or more of these treatment aspects.

The terms "recurrence," "relapse," or "relapsed" refer to the return of cancer or disease after clinical evaluation that the disease is clear. A diagnosis of distant metastasis or local recurrence is considered a relapse.

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

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 related to the stated purpose.

The term "therapeutically effective amount" refers to an amount of a caTCR or a composition comprising a caTCR as disclosed herein that is effective to "treat" a disease or condition in an individual. 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 cancer-related symptoms to some extent. To the extent that a caTCR or a composition comprising a caTCR as disclosed herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that 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 one or more symptoms associated with infection to some extent. In some embodiments, the therapeutically effective amount is an amount that prolongs the patient's survival.

As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is biologically or otherwise non-adverse, that is, the material can be incorporated into a pharmaceutical composition for administration to a patient without causing any significant adverse biological effect or interacting in a deleterious manner with any other component of the composition in which it is contained. Pharmaceutically acceptable carriers or formulations preferably meet the necessary standards for toxicology and manufacturing testing and/or are included in the Inactive Ingredient Guide established by the U.S. Food and Drug Administration.

It should be understood that the embodiments of the present invention described herein include "consisting of" and/or "consisting essentially of" the embodiments.

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

As used herein, reference to "not a value or parameter" generally means and describes "except for a value or parameter." For example, a method not intended for treating type X cancer means that the method is intended for treating cancers of types other than type X.

As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise.

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 invention, 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 the immunology, medicine, drugs and medicinal chemistry, and cell biology described herein are well known and commonly used in this art. In addition, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms. Throughout the specification and examples, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" should be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although various documents are cited herein, this citation does not constitute an admission that any of these documents forms 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 values similar to the stated reference value. In certain embodiments, unless otherwise stated 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 or less than) of the stated reference value. II. Expressing Constructs

The present invention provides one or more expression constructs, which include one or more expression cassettes, which are used to express caTCR, CSR and c-Jun polypeptide according to the caTCR (e.g., caTCR, which includes an antigen binding module that specifically binds to GPC3), CSR (e.g., CSR including a ligand binding module that can bind or interact with GPC3) and c-Jun polypeptide (e.g., human c-Jun polypeptide) described herein (the (one or more) expression constructs are also referred to herein as "(one or more) caTCR+CSR+c-Jun expression constructs"). 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). It should be understood that the terms "express" or "expressing" and "encode" or "encoding" are used interchangeably herein when used to describe a representation structure or a representation cartridge.

In some aspects, provided herein is one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 caTCR comprising: i) an antigen binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; CSR, comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a synergistic immune cell signaling module capable of providing a synergistic immune cell signaling module to the immune cell, wherein the ligand binding module and the synergistic immune cell signaling module are not derived from the same molecule, and wherein the anti-GPC3 CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the expression 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 multi-cistronic 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/δ TCR. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module, which comprises a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains 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 synergistic 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, provided herein is one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding module comprising: (1) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20; and (2) a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising an amino acid sequence of DDS, and _an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding module comprising: (1) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14; and (2) a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising an amino acid sequence of YDS, and an LC- _CDR3 comprising an amino acid sequence of SEQ ID NO: 17 ; ii) a transmembrane module; and iii) a synergistic immune cell signaling module capable of providing a synergistic stimulatory signal to the immune cell, wherein the ligand binding module and the synergistic immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3 caTCR comprises a _VH comprising an amino acid sequence of SEQ ID NO: 28 and a _VL comprising an amino acid sequence of SEQ ID NO: 29. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 30 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 10 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have 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 _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises an 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 synergistic 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.

In some embodiments, provided herein is one or more expression constructs comprising one or more expression cassettes for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding module comprising a scFv that is capable of binding or interacting with GPC3; ii) a transmembrane module derived from CD30; and iii) a co-stimulatory immune cell signaling module derived from CD30 that is capable of providing co-stimulatory signals to immune cells, wherein the anti-GPC3 CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the Fab comprises a heavy chain variable domain ( _VH ) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain ( _VL ) comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-GPC3 CSR comprises a fragment of CD30. In some embodiments, the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

In other aspects, provided herein is a multi-cistronic expression construct comprising an expression cassette for expressing: a) an anti-GPC3 caTCR comprising: i) an antigen binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human γ/δ TCR; b) an anti-GPC3 CSR comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a synergistic 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 NOs: 28 and 29, and the 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 NOs: 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 NOs: 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 NOs: 28 and 29, and a coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 1, a coding sequence of SEQ ID NOs: 30 and 31, and a coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 1, a coding sequence of SEQ ID NOs: 10 and 5, and a coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 1, a coding sequence of SEQ ID NOs: 30 and 31, and a coding sequence of SEQ ID NO: 16. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 1, a coding sequence of SEQ ID NOs: 10 and 5, and a coding sequence of SEQ ID NO: 16. In some embodiments, the expression cassette further comprises a coding sequence of a signal peptide. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 2. In some embodiments, the coding sequences are 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 the EF-1α promoter. In some embodiments, the construct is a viral vector (such as a lentiviral vector). A. Chimeric Antibody T Cell Receptor (caTCR) Construct

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 module 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 are capable of recruiting at least one TCR-associated signaling molecule (such as CD3δε, CD3γε, and/or ζζ). In some embodiments, the anti-GPC3 caTCR comprises an antigen binding module 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 module that specifically binds to GPC3; and b) a TCR module (TCRM) comprising a first TCR domain (TCRD) and a second TCRD, wherein the first TCRD comprises 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), and the second TCRD comprises 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 module is linked to the first and/or second TCRD. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity to each other that stabilizes the anti-GPC3 caTCR.

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 components of the anti-GPC3 caTCRs described herein (e.g., antigen binding module, 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.

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 more) times greater than its binding affinity for other molecules; or b) _{a Kd} that is no more than about 10 (such as no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000, or less) times greater than its Kd for binding to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). _Kd 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.

In some embodiments, the stabilization module comprises a first stabilization domain and a second stabilization domain. In some embodiments, the first and second stabilization domains have binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilization module is located between the antigen binding module and the TCRM. In some embodiments, the stabilization module is derived from the antibody portion. In some embodiments, the stabilization module is derived from the antibody constant domain. In some embodiments, the stabilization module is derived from the antibody heavy chain constant domain. In some embodiments, the antibody heavy chain constant domain contained in the stabilization module is derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain, which is human as the case may be. In some embodiments, the antibody heavy chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions and/or deletions) compared to the sequence from which it is derived. In some embodiments, the antibody light chain constant domain contained in the stabilization module is derived from a κ or λ light chain, whichever is human. In some embodiments, the stabilization module is derived from an antibody light chain constant domain. In some embodiments, the antibody light chain constant domain contained in the stabilization module is a variant comprising one or more modifications (e.g., amino acid substitutions, insertions and/or deletions) compared to the sequence from which it is derived. In some embodiments, the first and/or second stabilization domains comprise one or more modifications that do not substantially 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., a _CH3 domain) comprising a knob-into-hole modification. In some embodiments, the stabilizing module comprises an antibody constant domain region modified by electrostatic switching 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 and second stabilizing domains are linked by a disulfide bond.

Examples of stabilizing domains include an Fc region; a hinge region; a _CH2 domain; a _CH3 domain; a _CH4 domain; a _CH1 domain or a _CL domain; a TCR homeostatic domain; a leucine zipper domain (e.g., a jun/fos leucine zipper domain, see, e.g., Kostelney et al., J. Immunol , 148: 1547-1553, 1992; or a yeast GCN4 leucine zipper domain); an isoleucine zipper domain; a dimerization domain of a dimerizing cell surface receptor (e.g., interleukin-8 receptor (IL-8R); or an integrin heterodimer such as LFA-1 or GPIIIb/IIIa); a dimerization domain of a secreted dimerizing ligand (e.g., 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 (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., from about one, two or three to about ten cysteine residues), such that a disulfide bond(s) can be formed between the polypeptide and a second polypeptide comprising at least one cysteine residue.

In some embodiments, the TCRM described herein comprises a first T cell receptor domain (TCRD) and a second TCRD, the first TCRD comprising a first TCR transmembrane domain (TCR-TM), the second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-related signaling 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 αβ TCR or γδ TCR), 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, enhancing the recruitment of at least one TCR-related signaling molecule. The recruitment of TCR-related signaling molecules can be determined by methods known in the art, such as FACS analysis of surface expression of the TCR-CD3 complex or co-immunoprecipitation of CD3 subunits and caTCR. For example, in some embodiments, the first TCR-TM of the TCRRM described herein comprises, consists essentially of, or consists of the transmembrane domain of the TCR α chain (e.g., GenBank Accession No.: CCI73895) or a variant thereof, and the second TCR-TM of the TCRRM comprises, consists essentially of, or consists of the transmembrane domain of the TCR β chain (e.g., GenBank Accession No.: CCI73893) or a variant thereof. In some embodiments, the first TCR-TM comprises, consists essentially of, or consists of a transmembrane domain of a TCR δ chain (e.g., GenBank Accession No.: AAQ57272) or a variant thereof, and the second TCR-TM comprises, consists essentially of, or consists of a transmembrane domain of a TCR γ chain (e.g., GenBank Accession No.: AGE91788) or a variant thereof. In some embodiments, the first and second TCR-TMs of the TCRRMs described herein comprise, consist essentially of, or consist of a transmembrane domain of a constant domain of a TCR α chain (e.g., SEQ ID NO: 35) or a variant thereof and a transmembrane domain of a constant domain of a TCR β chain (e.g., SEQ ID NO: 36) or a variant thereof, respectively. In some embodiments, the first and second TCR-TMs respectively comprise, consist essentially of, or consist of: 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. In some embodiments, the first and second TCR-TMs respectively comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 39 and 40 or variants thereof. In some embodiments, the first and second TCR-TMs respectively comprise, consist essentially of, or consist of the amino acid sequences of SEQ ID NOs: 41 and 42 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 the reference sequence.

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 respectively comprise, consist essentially of, or consist of all or part of a connecting peptide of a TCR alpha chain constant domain, or a variant thereof, and all or part of a connecting peptide of a TCR beta chain constant domain, or a variant thereof. In some embodiments, the first and/or second connecting peptides comprise, consist essentially of, or consist of, respectively, all or a portion of a connecting peptide of a TCR δ chain constant domain or a variant thereof and all or a portion of a connecting peptide of a TCR γ chain constant domain or a variant thereof. In some embodiments, the first TCRD further comprises a first TCR intracellular domain at the carboxyl terminus of the first TCR-TM and/or the second TCRD further comprises a second TCR intracellular domain at the carboxyl terminus of the second TCR-TM. In some embodiments, the first TCR intracellular domain comprises all or a portion of an intracellular domain of a TCR subunit from which the first TCR-TM is derived or a variant thereof, and/or the second TCR intracellular domain comprises all or a portion of an intracellular domain of a TCR subunit from which the second TCR-TM is derived or a variant thereof. In some embodiments, the first TCRD is a fragment of one chain of a naturally occurring TCR or a variant thereof, and/or the second TCRD is a fragment of another chain of a naturally occurring TCR or a variant thereof. In some embodiments, at least one of the TCRDs is non-naturally occurring. The non-naturally occurring TCR domain may be the corresponding domain of a naturally occurring TCR, which has been 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.

In some embodiments, the first TCR-TM comprises, consists essentially of, or consists of SEQ ID NO: 3 or comprises all or a portion of 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: 3. In some embodiments, the second TCR-TM comprises, consists essentially of, or consists of SEQ ID NO: 4 or comprises all or a portion of 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: 4. In some embodiments, the first and second TCRDs are linked by a disulfide bond. In some embodiments, the first and second TCRDs are linked by a disulfide bond between a residue in the first linking peptide and a residue in the second linking peptide. In some embodiments, the 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, the TCRM is capable of recruiting each of CD3δε, CD3γε, and ζζ to form a caTCR-CD3 complex (i.e., promoting caTCR-CD3 complex formation).

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.

In some embodiments, the antigen binding moiety comprises a first antigen binding domain comprising an anti-GPC3 _VH antibody domain and a second antigen binding domain comprising an anti-GPC3 _VL antibody domain. In some embodiments, the anti-GPC3 _VH antibody domain and the anti-GPC3 _VL antibody domain CDRs are derived from the same antibody portion. In some embodiments, some of the anti-GPC3 _VH antibody domain and the anti-GPC3 _VL antibody domain CDRs are derived from different antibody portions. In some embodiments, the anti-GPC3 _VH antibody domain and/or the anti-GPC3 _VL antibody domain are human antibody domains, humanized antibody domains, chimeric antibody domains, semi-synthetic antibody domains, or fully synthetic antibody domains.

In some embodiments, the antigen binding moiety is an antibody portion comprising specific CDR sequences derived from one or more antibody portions specific for GPC3 (such as monoclonal antibodies) or certain variants of such sequences comprising 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 properties (such as specificity for and/or cross-reactivity with related variants of GPC3) are also encompassed.

In some embodiments, the anti-GPC3 caTCR comprises an antigen binding module described herein, optionally including a stabilization module, linked to a TCRM described herein. For example, in some embodiments, the anti-GPC3 caTCR comprises an antigen binding module linked to the N-terminus of one or both of the TCRDs. In some embodiments, the anti-GPC3 caTCR comprises a stabilization module between the TCRM and the antigen binding module. In some embodiments, the anti-GPC3 caTCR further comprises a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers between about 5 to about 70 (e.g., any of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any range between such values) amino acids in length. In some embodiments, the anti-GPC3 caTCR further comprises one or more accessory intracellular domains. In some embodiments, the one or more accessory intracellular domains are located at the carboxyl terminus of the first TCRD and/or the second TCRD. In some embodiments, the one or more accessory 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, the one or more accessory intracellular domains individually comprise TCR co-stimulatory domains. In some embodiments, the TCR co-stimulatory domain comprises all or a portion of the intracellular domain of an immune co-stimulatory molecule (e.g., CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like).

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 a second TCR-TM derived from a transmembrane domain of 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 linked to the amino terminus of one or more polypeptide chains in the TCRM. For example, in some embodiments, the TCRM comprises two polypeptide chains, and the antigen binding module is linked to the amino terminus of one or both of the TCRM polypeptide chains. In some embodiments, the first and second TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, the first and second TCR-TMs are non-naturally occurring. In some embodiments, TCRM further comprises at least one connecting peptide or fragment thereof of TCR at the amino terminal of TCR-TM. In some embodiments, TCRM further comprises at least one TCR intracellular domain, and the at least one TCR intracellular domain comprises a sequence from the TCR intracellular domain at the carboxyl terminal of TCR-TM. In some embodiments, TCRM comprises a TCRD derived from a fragment of the 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 synergistic stimulation signaling sequence (such as from CD27, CD28, 4-1BB (CD137), OX40, CD30 or CD40) at the carboxyl terminal of TCR-TM. In some embodiments, caTCR lacks a synergistic stimulation 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 an anti-GPC3 _VH antibody domain and an anti-GPC3 _VL antibody domain. In some embodiments, the anti-GPC3 antibody portion is a human antibody domain, a humanized antibody domain, a chimeric antibody domain, a semi-synthetic antibody domain, or a fully synthetic antibody domain. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization moiety comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the stabilization moiety comprises at least one disulfide bond connecting the stabilization domains. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C _H 1 and _CL antibody domains) or variants thereof. In some embodiments, the 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, the TCRM allows enhanced recruitment of at least one TCR-related signaling molecule compared to a TCRM comprising a T cell receptor transmembrane domain. In some embodiments, the 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 anti-GPC3 caTCR is a heteropolymer, such as a heterodimer. For example, 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, wherein the antigen binding module is linked to the first and/or second polypeptide chain.

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 the TCR; 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 linked 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 αβ TCR and the first and second TCR-TMs are derived from the TCR α and β subunit transmembrane domains. In some embodiments, the TCR is a γδ TCR, and the first and second TCR-TMs are derived from TCR γ 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 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 linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR intracellular domain, and/or the second TCRD further comprises a second TCR intracellular domain. In some embodiments, the first TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the first TCR-TM is derived, and/or the second TCR intracellular domain comprises a sequence from the intracellular domain of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of the TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of the TCR subunit from which the second TCR-TM is derived. In some embodiments, the anti-GPC3 caTCR further comprises at least one accessory intracellular domain comprising a T cell co-stimulatory signaling sequence (e.g., 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 and second stabilizing domains have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first and second stabilizing domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C _H 1 and _CL antibody domains) or variants thereof. In some embodiments, the 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, the TCRM allows enhanced recruitment of at least one TCR-related signaling molecule compared to a TCRM comprising a T cell receptor transmembrane domain. In some embodiments, the TCRM promotes caTCR-CD3 complex formation. In some embodiments, there is a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the antigen binding module is an antibody 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).

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 γδ TCR; and a second TCRD comprising a second TCR-TM derived from another transmembrane domain of a naturally occurring γδ 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 GPC3a, wherein the antigen binding module is linked 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-linking peptide or a fragment thereof, and/or the second TCRD further comprises a second TCR-linking 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 linked by a disulfide bond. In some embodiments, the first TCRD further comprises a first TCR intracellular domain, and/or the second TCRD further comprises a second TCR intracellular domain. In some embodiments, the first TCR intracellular domain comprises a sequence from an intracellular domain of a TCR subunit from which the first TCR-TM is derived, and/or the second TCR intracellular domain comprises a sequence from an intracellular domain of a TCR subunit from which the second TCR-TM is derived. In some embodiments, the first TCRD is a fragment of a TCR subunit from which the first TCR-TM is derived, and/or the second TCRD is a fragment of a TCR subunit from which the second TCR-TM is derived. In some embodiments, the anti-GPC3 caTCR further comprises at least one auxiliary intracellular domain comprising a T cell co-stimulatory signaling sequence (e.g., from CD27, CD28, 4-1BB (CD137), OX40, CD30, or CD40). In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity to each other that stabilizes the anti-GPC3 caTCR. In some embodiments, the first and second stabilization domains are linked by a disulfide bond. In some embodiments, the first and second stabilizing domains comprise antibody domains (such as C _H 1 and _CL antibody domains) or variants thereof. In some embodiments, the 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, the TCRM allows enhanced recruitment of at least one TCR-related signaling molecule compared to a TCRM comprising a naturally occurring γδ T cell receptor transmembrane domain. In some embodiments, the TCRM promotes caTCR-CD3 complex formation. In some embodiments, there is a spacer module between any two anti-GPC3 caTCR modules or domains. In some embodiments, the antigen binding module is an antibody 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).

In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC-CDR1 comprising an 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 or variants thereof, a 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 a VL comprising the amino acid sequence of SEQ ID NO: 11 or a LC-CDR3 comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or _variants thereof. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a _VL 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. In some embodiments, the antigen binding moiety is a Fab.

In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC-CDR1 comprising an 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 or variants thereof, a LC-CDR2 comprising the amino acid sequence of YDS or variants thereof comprising up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions, and a _VL comprising the amino acid sequence of SEQ ID NO: 17 or LC-CDR3 comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or variants thereof. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14, and a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15, an LC-CDR2 comprising an amino acid sequence of YDS, and an LC-CDR3 comprising an amino acid sequence of _SEQ ID NO: 17. In some embodiments, the antigen binding moiety is a Fab.

In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC-CDR1 comprising an 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 or variants thereof, a 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 a VL comprising the amino acid sequence of SEQ ID NO: 23 or a LC-CDR3 comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or _variants thereof. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding module comprising: a VH comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a _VL 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. In some embodiments, the anti-GPC3 antibody portion is Fab.

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

In some embodiments, an anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a _VL comprising an amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID _NO : 27. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a) a _VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: an HC-CDR of _{a VH} comprising the amino acid sequence of SEQ ID NO: 26, and an LC-CDR of _{a VL} comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antigen binding moiety is a Fab.

In some embodiments, an anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a _VL comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID _NO : 29. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: a) a _VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a _VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-GPC3 caTCR comprises an antigen binding moiety comprising: an HC-CDR of _{a VH} comprising the amino acid sequence of SEQ ID NO: 28, and an LC-CDR of _{a VL} comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antigen binding moiety is a Fab.

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 _VH fused to a TCRD derived from a TCR α chain and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR β chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a _VH fused to a TCRD derived from a TCR β chain and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR α chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a _VH fused to a TCRD derived from a TCR delta chain, and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR gamma chain. In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising a first polypeptide chain comprising a _VH fused to a TCRD derived from a TCR gamma chain, and a second polypeptide chain comprising a _VL fused to a TCRD derived from a TCR delta chain. As used herein, the term "fused" means direct fusion or indirect fusion.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 66.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the anti-GPC3 caTCR is a heterodimer comprising: i) a first polypeptide chain comprising an 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 an 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) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70. B. Chimeric Co-stimulatory Receptor (CSR) Constructs

The expression constructs provided herein further encode a chimeric co-stimulatory receptor (CSR) that specifically binds 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 synergistic stimulatory signal transduction 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.

Examples of synergistic immune cell signaling domains used in the anti-GPC3 CSR 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 after caTCR engagement; and any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities.

In some cases, the signal generated by the TCR alone is insufficient to fully activate T cells, and co-stimulatory signals are also required. Thus, in some embodiments, T cell activation is mediated by two distinct classes 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 co-stimulatory signals (referred to herein as "co-stimulatory T cell signaling sequences").

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, which may include 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.

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.

The target 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.

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 ( _VH and/or _VL domains) of an antibody portion that is specific for GPC3.

In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC-CDR1 comprising an 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 or variants thereof, a 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 a VL comprising the amino acid sequence of SEQ ID NO: 11 or a LC-CDR3 comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or _variants thereof. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a _VL 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. In some embodiments, the ligand binding module is a scFv.

In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC- _CDR1 comprising an 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 or variants thereof, a LC-CDR2 comprising the amino acid sequence of YDS or variants thereof comprising up to about 3 (such as any one of about 1, 2 or 3) amino acid substitutions, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or _VL comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or variants thereof. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH 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, and a _VL 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. In some embodiments, the ligand binding module is a scFv.

In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising an HC-CDR1 comprising an 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 an 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 an 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, and a LC-CDR1 comprising an 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 or variants thereof, a 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 a VL comprising the amino acid sequence of SEQ ID NO: 23 or a LC-CDR3 comprising up to about 5 (such as any one of about 1, 2, 3, 4 or 5) amino acid substitutions or _variants thereof. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, and a _VL 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. In some embodiments, the ligand binding module is a scFv.

In some embodiments, an anti-GPC3 CSR comprises a ligand binding module comprising: a VH comprising an amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 24, and a _VL comprising an amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: ₂₅ . In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a) a _VH comprising the amino acid sequence of SEQ ID NO: 24; and b) a _VL comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a HC-CDR of _{a VH} comprising the amino acid sequence of SEQ ID NO: 24, and a LC-CDR of _{a VL} comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the ligand binding module is a scFv.

In some embodiments, an anti-GPC3 CSR comprises a ligand binding module comprising: a VH 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 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 26, and a _VL comprising an amino acid sequence of SEQ ID NO: 27, 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: 27. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a) a _VH comprising the amino acid sequence of SEQ ID NO: 26; and b) a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a HC-CDR of _{a VH} comprising the amino acid sequence of SEQ ID NO: 26, and a LC-CDR of _{a VL} comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the ligand binding module is a scFv.

In some embodiments, an anti-GPC3 caTCR comprises an antigen binding moiety comprising: a VH comprising an amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 28, and a _VL comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that has at least about 80% (including, for example, any one of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID _NO : 29. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a) a _VH comprising the amino acid sequence of SEQ ID NO: 28; and b) a _VL comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the anti-GPC3 CSR comprises a ligand binding module comprising: a HC-CDR of _{a VH} comprising the amino acid sequence of SEQ ID NO: 28, and a LC-CDR of _{a VL} comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the ligand binding module is a scFv.

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) to SEQ ID NO: 50. In some embodiments, the anti-GPC3 CSR comprises a transmembrane domain derived from the transmembrane domain of CD8. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 or 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: 22.

In some embodiments, the anti-GPC3 CSR co-stimulatory immune cell signaling module is derived from the intracellular domain of the co-stimulatory receptor of the TCR. In some embodiments, the anti-GPC3 CSR co-stimulatory immune cell signaling module comprises, consists essentially of, or consists of, for example, all or a portion of the intracellular domain of a co-stimulatory 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 that specifically binds to CD83, and the like. In some embodiments, the anti-GPC3 CSR's synergistic stimulatory immune cell signaling module comprises, consists essentially of, or consists of all or a portion of the intracellular domain of a synergistic stimulatory receptor for CD30, CD28, 4-IBB, OX40, ICOS, CD27, or CD40. In some embodiments, the synergistic stimulatory immune cell signaling module of the CSR is derived from human CD30. In some embodiments, the synergistic stimulatory 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.

In some embodiments, the synergistic stimulatory immune cell signaling module of CSR is derived from human CD28. In some embodiments, the synergistic stimulatory immune cell signaling module derived from human CD28 comprises an 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 synergistic stimulatory immune cell signaling module derived from human CD28 comprises an 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) to SEQ ID NO: 46. In some embodiments, the synergistic stimulatory immune cell signaling module of CSR is derived from human 4-IBB. In some embodiments, the synergistically stimulatory immune cell signaling module derived from human 4-IBB comprises an 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) to SEQ ID NO: 47. In some embodiments, the synergistically stimulatory immune cell signaling module derived from human 4-IBB comprises an 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) to SEQ ID NO: 48.

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 synergistic stimulatory signal transduction 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 any one of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length.

In some embodiments, the ligand binding moiety (e.g., 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 more) times greater than its binding affinity for other molecules; or b) _{a Kd} that is more than about 1/10 (e.g., no more than about any of 1/10, 1/20, 1/30, 1/40, 1/50, 1/75, 1/100, 1/200, 1/300, 1/400, 1/500, 1/750, 1/1000, or less) times greater than its Kd for binding to other molecules. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation analysis (RIA). _Kd 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.

In some embodiments, the anti-GPC3 CSR described herein comprises a) scFv; and b) CD30 fragment. In some embodiments, the scFv comprises a VH domain 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, and a _VL 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. In some embodiments, the scFv comprises a _VH domain having an amino acid sequence of SEQ ID NO: 26, and _{a VL domain} having an amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 33. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the fragment of CD30 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 44 from the amino terminus to the carboxyl terminus. In some embodiments, the CSR comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least about 90% sequence identity (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16. In some embodiments, the anti-GPC3 CSR comprises the amino acid sequence of SEQ ID NO: 16.

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

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 (obtained in GenBank with accession number AAA59197.1 or in UniProtKB (with accession number P05412.2): See also Hattori et al., PNAS (1988) 85:9148-52.

In humans, the c-Jun protein is encoded by the JUN gene located on chromosome 1 (GenBank accession number NC_000001.11, nucleotides 58,780,791 to 58,784,047, in the negative 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 length of the wild-type human c-Jun protein sequence is 331 amino acids.

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.

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 functionally abnormal (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 epsilon, bZIP and amino acid C-terminus) of wild-type c-Jun. In some embodiments, the N-terminal amino acid residue (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 acid N-terminus) of wild-type c-Jun is deleted, mutated, or otherwise inactivated.

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

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.

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.

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 comprises a nucleic acid sequence listed in any of SEQ ID NOs: 52-62.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Exemplary c-Jun nucleotide sequences are provided in the Sequence Listing provided herein.

The codons of the c-Jun nucleotide sequences disclosed herein can be optimized using any method known in the art. For example, in some aspects, the codons of the c-Jun nucleotide sequences 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 fitness 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, dyad repeat sequences); (v) restriction enzyme recognition sites; or (vi) combinations thereof.

Although certain disclosures provided herein are generally directed 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 equally applicable to other methods (e.g., transcriptional activators, such as CRISPRa) for inducing and/or increasing c-Jun protein expression in cells provided herein.

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.

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

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

Assays suitable for measuring depletion, cell phenotype, persistence, cytotoxicity and/or killing, proliferation, interleukin production/release, and gene expression profiles are known in the art and include, for example, flow cytometry, intracellular interleukin staining (ICS), INCUCYTE® immunocytotoxicity assay, Meso Scale Discovery (MSD, a platform combining electrochemical luminescence with MULTI-ARRAY technology) or similar assays, persistence antigen stimulation assays, bulk and single cell RNAseq (see, e.g., Front Genet. 2020; 11:220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci. 2:139-173), cytotoxicity/killing assays, ELISA, Western blots, and other standard molecular and cell biology methods (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 analyses. D. Nucleic Acids

Anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun can be introduced into T cells or progenitor 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.

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 genomes of which contain 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.

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 cassette being its own transcriptional unit (e.g., with its own promoter and polyadenylation site and other transcriptional control elements). In specific embodiments, the four coding sequences can be placed in a single expression cassette (e.g., a polycistronic expression cassette), where the four coding sequences are transcribed under a common promoter. In a polycistronic arrangement, the coding sequences are in frame and separated from each other by 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, a polycistronic (e.g., tetracistronic) expression cassette is transcribed into a single RNA, but ultimately the single RNA is processed and translated into independent polypeptides.

In some embodiments, the polycistronic expression cassette comprises the coding sequence of anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun. In some embodiments, the expression cassette comprises the coding sequence of the antigen binding module 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 human γ/δ TCR. 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 NOs: 10 and 5. In some embodiments, the expression cassette comprises the coding sequence of the ligand binding module 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 the coding sequence of the partial sequence of human CD30 of the anti-GPC3 CSR. In some embodiments, the expression cassette comprises the coding sequence of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the expression cassette comprises the coding sequence of the human c-Jun polypeptide. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1. In some embodiments, the expression cassette comprises the coding sequence of SEQ ID NO: 1, the coding sequences of SEQ ID NOs: 28 and 29, and the 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 NOs: 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 NOs: 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 NOs: 28 and 29, and a coding sequence of SEQ ID NOs: 33 and 43. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 1, a coding sequence of SEQ ID NOs: 30 and 31, and a coding sequence of SEQ ID NOs: 33 and 43. 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 and 43. In some embodiments, the expression cassette further comprises a coding sequence of a signal peptide. In some embodiments, the expression cassette comprises a coding sequence of SEQ ID NO: 2.

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 Estein-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 encompassed. Alternatively, the expression cassette may comprise an inducible promoter system. Exemplary inducible promoter systems include, but are not limited to, hormone regulatory elements, synthetic ligand regulatory elements, ionizing radiation regulatory elements, 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 comprises a signal peptide. In some embodiments, the signal peptide comprises a coding sequence for the amino acid sequence of SEQ ID NO: 2.

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

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.

In certain embodiments, the present invention provides a single vector construct (e.g., a lentiviral vector) comprising a multi-cistronic expression cassette comprising a mammalian promoter, a c-Jun coding sequence, a coding sequence for two anti-GPC3 caTCR chains, a coding sequence for an anti-GPC3 CSR, and a polyadenylation signal sequence. The coding sequences are connected by a nucleotide linker, which can 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, FIG. 1A illustrates such an expression cassette, wherein the promoter is the EF-1α promoter.

In a specific embodiment, the expression cassette encodes 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., construct 2 in FIG. 1A ). The construct may be a recombinant lentiviral vector and may further comprise a central polypurine tract (cPPT) upstream of the EF-1α promoter and a WPRE sequence between the CSR coding sequence and the SV40 polyadenylation signal or other sequences that are effectively transduced and expressed in mammalian cells.

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

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. Integration can be targeted integration via gene editing (e.g., mediated by CRISPR, TALEN, zinc finger nucleases and meganucleases).

The 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, a flow cytometry analysis. To confirm c-Jun expression, RT-PCR can be performed on the engineered T cells. Positive selection can result in an enrichment of caTCR ⁺ CSR ⁺ c-Jun ⁺ cells in a cell population, 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 cryogenically stored until use. E. Variants

In some embodiments, amino acid sequence variants of the anti-GPC3 caTCR, anti-GPC3 CSR, and c-Jun provided herein are encompassed. In some embodiments, amino acid sequence variants of the antigen binding module of the anti-GPC3 caTCR and/or the ligand binding module of the anti-GPC3 CSR are encompassed. 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). The amino acid sequence variants of the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-GPC3 caTCR, anti-GPC3 CSR, and/or c-Jun or by peptide synthesis. Such modifications include, for example, deletion and/or insertion and/or substitution 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 may be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as GPC3 binding.

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 substitutional mutagenesis include hypervariable regions (HVRs) and framework regions (FRs).

Conservative substitutions are shown in Table 2 below. Table 2. Conservative substitutions . Original Residue Exemplary substitutions Better replacement 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; nor-leucine Leu Leu (L) nor-leucine; 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; nor-leucine Leu

Amino acids can be divided into different categories based on common side chain properties: a. Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: 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.

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

Exemplary substitution variants are affinity matured antibody portions consisting of an antigen binding module of an anti-GPC3 caTCR or a ligand binding module of an anti-GPC CSR, which can be conveniently generated, for example, using affinity maturation techniques based on phage display. Briefly, 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 the HVRs, for example, to improve antibody portion affinity. Such changes can be made in HVR "hotspots," i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or specificity determining residues (SDRs), where the resulting variant _VH or _VL is tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from the secondary libraries has been described, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)).

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 induction). 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-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, for example using alanine scanning mutagenesis induction or modeling. CDR-H3 and CDR-L3 are often particularly targeted.

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 GPC3. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in 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.

A useful method for identifying residues or regions of an antibody (e.g., the antigen binding module of an anti-GPC3 caTCR or the portion of an antibody contained in an anti-GPC3 CSR) that can be targeted for mutation induction is called "alanine scanning mutation induction," as described by Cunningham and Wells (1989) Science , 244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (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 substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be determined to identify the 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 substitution candidates. Variants can be screened to determine whether they exhibit the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibody portions (e.g., antigen binding moieties of anti-GPC3 caTCRs and/or ligand binding moieties of anti-GPC3 CSRs) with an N-terminal methionyl residue. Other insertion variants of the antibody portion include fusions of the N- or C-terminus of the antibody portion with an enzyme (e.g., for use in 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

In some embodiments, the anti-GPC3 caTCR, anti-GPC3 CSR and/or c-Jun provided herein are altered to increase or decrease the degree of glycosylation of the expression construct. Addition or deletion of glycosylation sites of 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 polypeptide portion thereof to create or remove one or more glycosylation sites.

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 linked to Asn297 of the CH2 domain of the Fc region, generally by an N-linkage (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-GPC3 constructs of the present invention may be modified to produce anti-GPC3 construct variants with improved properties.

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 ability of Fc to FcγRIIIa. Enhanced FcγRIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications requiring cytotoxicity.

In some embodiments, when Fc-mediated cytotoxicity is not desired, the 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

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 those residues with cysteine, reactive thiol groups are located at accessible sites of the construct and can be used to conjugate the anti-GPC3 construct to other moieties. Cysteine engineered expression constructs can be generated, for example, as described in U.S. Patent No. 7,521,541. III. Immune Cells

In one aspect, the present 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 for 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.

In some embodiments, the present invention provides an immune cell (such as a T cell) that presents 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 the 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 αβ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR δ and γ chains, or the T cell is a γδ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of the endogenous TCR subunits of the immune cell. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of TCR γ and/or δ chain. Cellular modification for disrupting gene expression includes any such techniques known in the art, including, for example, RNA interference (e.g., siRNA, shRNA, miRNA), gene editing (e.g., CRISPR or TALEN-based gene knockout) and the like.

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 by 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 an anti-GPC3 caTCR, a second caTCR nucleic acid sequence encoding a second caTCR polypeptide chain of an anti-GPC3 caTCR, an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of an 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 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 an 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 promoters have different sequences. In some embodiments, some or all nucleic acid sequences are under the control of a single promoter. In some embodiments, some or all promoters are inductive. 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.

Thus, in some embodiments, an anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun ("caTCR+CSR+c-Jun") immune cell (e.g., 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 encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; and d) a c-Jun nucleic acid sequence encoding a c-Jun polypeptide, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed by 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 by the anti-GPC3 CSR nucleic acid to form an anti-GPC3 CSR, wherein the c-Jun polypeptide is expressed by 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 immune cells. 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 inductive. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors). In some embodiments, the immune cells do not express the TCR subunits of the TCR-TM from which the caTCR is derived. For example, in some embodiments, the immune cell is an αβ T cell and the introduced TCR-TM of the anti-GPC3 caTCR comprises sequences derived from the TCR δ and γ chains, or the immune cell is a γδ T cell and the introduced TCR-TM of the anti-GPC3 caTCR comprises sequences derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of the TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of the TCR γ and/or δ chain. 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. In some embodiments, some or all of the vectors are viral vectors (such as lentiviral vectors) integrated into the host genome of the immune cells.

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 of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a human c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second vector comprising a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR. c) a third vector comprising a third promoter operably linked to an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain; 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 by the first and second anti-GPC3 caTCR nucleic acid sequences to form an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed by the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed by 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 inductive. 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 αβ T cell and the TCR-TM of the introduced caTCR includes a sequence derived from the TCR δ and γ chains, or the immune cell is a γδ T cell and the TCR-TM of the introduced caTCR includes a sequence derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of TCR γ 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) integrated into the host genome of the immune cell.

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 of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first promoter operably linked to a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; and ii) a second promoter operably linked to a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR. caTCR nucleic acid sequence; b) a second vector comprising a third promoter operably linked to an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain; 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 by the first and second anti-GPC3 caTCR nucleic acid sequences to form an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed by the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed by the c-Jun nucleic acid sequence, and wherein the anti-GPC3 caTCR is 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 inductive. 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 αβ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from the TCR δ and γ chains, or the immune cell is a γδ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of TCR γ 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) integrated into the host genome of the immune cell. It will be appreciated that embodiments are also contemplated in which either of the nucleic acid sequences are exchanged, 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.

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 of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a) a first vector comprising i) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, and ii) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR, wherein the first and second anti-GPC3 The invention relates to a method for expressing an anti-GPC3 caTCR polypeptide chain in an immune cell. The method comprises the following steps: a) a first promoter, wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed by the first and second anti-GPC3 caTCR nucleic acid sequences to form an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed by the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed by the c-Jun nucleic acid sequence, and 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 link 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, the first promoter is operably linked to the 5' end of the second anti-GPC3 caTCR nucleic acid sequence, and there is 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) connecting 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 cell does not express the TCR subunits from which the TCR-TM of the caTCR is derived. For example, in some embodiments, the immune cell is an αβ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR δ and γ chains, or the immune cell is a γδ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of TCR γ 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) integrated into the host genome of the immune cell. It will be appreciated that embodiments are also contemplated in which either of the nucleic acid sequences are exchanged, 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.

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 of the caTCRs described herein, an anti-GPC3 CSR according to any of the CSRs described herein, and a c-Jun polypeptide according to any of the c-Jun polypeptides described herein, wherein the caTCR+CSR+c-Jun immune cell comprises a vector comprising: a) a first anti-GPC3 caTCR nucleic acid sequence encoding a first anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; b) a second anti-GPC3 caTCR nucleic acid sequence encoding a second anti-GPC3 caTCR polypeptide chain of the anti-GPC3 caTCR; c) an anti-GPC3 CSR nucleic acid sequence encoding an anti-GPC3 CSR polypeptide chain of the anti-GPC3 CSR; CSR nucleic acid sequence; 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 CSR nucleic acid sequence and the c-Jun polypeptide are under the control of a single promoter; wherein the first and second anti-GPC3 caTCR polypeptide chains are expressed by the first and second anti-GPC3 caTCR nucleic acid sequences to form an anti-GPC3 caTCR, the anti-GPC3 CSR polypeptide chain is expressed by the anti-GPC3 CSR nucleic acid sequence to form an anti-GPC3 CSR, the c-Jun polypeptide is expressed by 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 by nucleic acid linkers individually 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), 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-GPC3 caTCR is derived. For example, in some embodiments, the immune cell is an αβ T cell and the TCR-TM of the introduced anti-GPC3 caTCR comprises sequences derived from the TCR δ and γ chains, or the immune cell is a γδ T cell and the TCR-TM of the introduced caTCR comprises sequences derived from the TCR α and β chains. In some embodiments, the immune cell is modified to block or reduce the expression of one or both of its endogenous TCR subunits. For example, in some embodiments, the immune cell is an αβ T cell modified to block or reduce the expression of the TCR α and/or β chain, or the immune cell is a γδ T cell modified to block or reduce the expression of the TCR γ 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) integrated into the host genome of the immune cell. A. Source of immune cells

The source of the engineered immune cells of the present invention can be the patient to be treated (i.e., autologous cells) or from a donor that is not the patient to be treated (e.g., allogeneic cells). In some embodiments, the engineered immune cells are engineered T cells. The engineered T cells herein 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 a mixture thereof. The T cells to be engineered can be autologous or allogeneic.

Prior to T cell expansion and genetic modification, T cells are obtained from an individual. 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 the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the present invention, any number of T cell strains available in this technology can be used. In some embodiments of the present invention, T cells can be obtained from blood units collected from an individual using any number of techniques known to those skilled in the art (such as Ficoll™ separation). In some embodiments, cells from the circulating blood of an individual are obtained by hemacytesis. The product of hemopheresis generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by hemopheresis can be washed to remove the plasma fraction and the cells are placed in an appropriate buffer or 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 appreciated by those of ordinary skill in the art, the washing step can be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automated "flow-through" centrifuge (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 Ca2 ⁺ -free, Mg2 ⁺ -free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the hemocytosis sample can be removed and the cells resuspended directly in culture medium.

In some embodiments, T cells are separated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by countercurrent centrifugal elutriation. Specific subsets of T cells, such as CD3 ⁺ , CD28 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ , and CD45RO ⁺ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubating with anti-CD3/anti-CD28 (i.e., 3×28) conjugated beads, such as ^DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient for positive selection of the desired T cells. 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 cultivation time period is 24 hours. In order to separate T cells from leukemia patients, using a longer cultivation time (such as 24 hours) can improve cell yield. In any case where T cells are few compared to other cell types, a longer cultivation time can be used to separate T cells, such as separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunodeficient individuals. In addition, the use of longer incubation times can increase the efficiency of capturing CD8 ⁺ T cells. Therefore, by shortening or extending the time that T cells are allowed to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected at the beginning of culture or at other time points during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be preferentially selected for or against at the beginning 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 the activation and expansion process. The "unselected" cells can also undergo additional rounds of selection.

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 approach is to sort and/or select cells by negative magnetic immunoadhesion or flow cytometry using a cocktail 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 for regulatory T 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.

To isolate a desired cell population by positive or negative selection, the cell concentration and surface (e.g., particles, such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume of beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. For example, in some embodiments, a concentration of about 2 billion cells/ml is used. In some embodiments, a concentration of about 1 billion cells/ml is used. In some embodiments, greater than about 100 million cells/ml is used. In some embodiments, a cell concentration of about 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In some embodiments, a cell concentration of about 75, 80, 85, 90, 95, or 100 million cells/ml is used. In some embodiments, a concentration of about 125 million or about 150 million cells/ml is used. Use of high concentrations can result in increased cell yield, cell activation, and cell expansion. Additionally, using high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells, or cells from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and be desirable to obtain. For example, using high cell concentrations allows for more efficient selection of CD8 ⁺ T cells, which typically have weak CD28 expression.

In some embodiments of the invention, T cells are obtained directly from a patient following self-treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatments with drugs that disrupt the immune system, T cells obtained may be of optimal quality or have improved ability to expand ex vivo shortly after treatment during a period when patients would normally recover from self-treatment. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a better state to enhance engraftment and in vivo expansion. Thus, the collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase is encompassed within the context of the invention. In addition, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning therapy can be used to establish conditions within an individual in which specific cell types are favored to reconstitute, recirculate, regenerate, and/or expand, particularly during a defined time window following treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Whether before or after genetically modifying T cells to express a desired expression construct, generally, methods such as those described in 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; 7,144,5 75; 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 the method described in U.S. Patent Application Publication No. 20060121005 to activate and expand T cells.

In general, the T cells of the present invention are expanded by contacting an agent that stimulates CD3/TCR complex-related signals and a ligand that stimulates a co-stimulatory molecule on the surface of the T cell with the surface to which it is attached. Specifically, the T cell population can be stimulated as follows: for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD3 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., lichen planus) that binds to a calcium ion carrier. In order to co-stimulate an accessory molecule on the surface of the T cell, a ligand that binds to the accessory molecule is used. For example, the T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate the proliferation of CD4 ⁺ T cells or CD8 ⁺ T cells, anti-CD3 antibodies and anti-CD28 antibodies can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), which can be used according to other methods generally known in the art (Berg et al., Transplant Proc . 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med . 190(9):13191328, 1999; Garland et al., J. Immunol. Meth . 227(1-2):53-63, 1999).

Cell culture conditions may include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, reagents, such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors, such as interleukins, 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.

In some embodiments, the cells to be engineered may be pluripotent or multipotent cells that differentiate into mature T cells after engineering. These non-T cells may be allogeneic and may be, for example, human embryonic stem cells, human induced pluripotent stem cells, or hematopoietic stem cells or pioneer cells. For ease of description, pluripotent and multipotent cells are collectively referred to herein as "pioneer cells."

In the case of using allogeneic cells, it is preferred that the allogeneic cells be engineered to reduce graft-versus-host rejection (e.g., by genetically deleting endogenous B2M and/or TRAC genes). B. Engineering of immune cells or progenitor cells

In some aspects, cell 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 cell exhibits increased c-Jun expression compared to a corresponding cell that has not been modified), or any combination thereof. As further described elsewhere herein, in some aspects, cells can be engineered or modified with a transcriptional activator (e.g., a transcriptional activator based on a CRISPR/Cas system), wherein the transcriptional activator is capable of inducing and/or increasing endogenous expression of a protein of interest (e.g., c-Jun).

In some aspects, the cells described herein have been modified with a transcriptional activator that is capable of inducing and/or increasing the endogenous expression of a protein of interest (e.g., c-Jun) in the cell. As used herein, the term "transcriptional activator" refers to a protein that increases the transcription of a gene or gene body (e.g., by binding to an enhancer or promoter proximal element 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 interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems, or combinations thereof. See, e.g., Kabadi et al., Methods (2014) 69(2):188-197, which is incorporated herein by reference in its entirety.

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, e.g., Pandelakis et al., Cell Systems (2020) 10(1): 1-14, which is incorporated herein by reference in its entirety). In some aspects, the modified Cas protein comprises a modified Cas9 protein (also referred to as "dCas9" in the art). In some aspects, the modified Cas protein comprises a modified Cas12a protein. In some aspects, a modified Cas protein suitable for use in the present invention is combined with a guide polynucleotide (e.g., a small guide RNA) ("modified Cas guide complex"), wherein the guide polynucleotide comprises a recognition sequence that is complementary to a region of a nucleic acid sequence encoding a protein of interest (e.g., c-Jun). In certain aspects, the guide polynucleotide comprises a recognition sequence that is complementary to 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 a modified Cas guide complex (e.g., the N-terminus and/or C-terminus of a 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 a regulatory element (e.g., a promoter region) of an 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).

In some aspects, one or more transcriptional repressors (e.g., Kruppel-associated box domain (KRAB)) can 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 can inhibit or reduce the transcription of genes, such as those genes (e.g., Bach2) that can interfere with c-Jun expression. 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 can be attached to both one or more transcriptional activators and one or more transcriptional repressors.

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) linked to a protease (e.g., tobacco etch virus (TEV)) and a single guide RNA (sgRNA) targeting the promoter region of c-Jun. In some aspects, the cell is modified to further 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.

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) exogenous nucleotide sequences encoding one or more proteins (e.g., anti-GPC3 caTCR, 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-GPC3 caTCR), (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 cell may further comprise an exogenous nucleotide sequence encoding a third protein (e.g., EGFRt). As described herein, in some aspects, the exogenous nucleotide sequences encoding the first, second, and third proteins may be part of a single polycistronic vector.

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

In some aspects, the cell has 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 a corresponding cell that has not been modified with the transcriptional activator.

Although certain disclosures provided herein are generally directed 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 herein using exogenous nucleotide sequences are equally applicable to other methods provided herein for inducing and/or increasing c-Jun protein expression in cells (e.g., transcriptional activators, such as CRISPRa).

The immune cells (e.g., T cells) or progenitor cells herein can be engineered to express anti-GPC3 caTCRs and anti-GPC3 CSRs, and overexpress c-Jun (e.g., human c-Jun polypeptides). Anti-GPC3 caTCRs can specifically bind to GPC3 tumor cells, and anti-GPC3 CSRs can specifically bind to GPC3 on tumor cells (e.g., the same GPC3, but different GPC3 antigenic determinants). As used herein, a receptor (e.g., anti-GPC3 caTCR or anti-GPC3 CSR) is said to specifically bind to GPC3 when _{the KD} of binding is ≤100 nM, e.g., ≤10 nM or ≤1 nM. The _KD binding affinity constant can be measured, for example, by surface plasmon resonance (using, for example, a Biacore™ or Octet™ system).

In some embodiments, the immune cells express anti-GPC3 caTCR with uniform cell surface distribution. In some embodiments, the immune cells express anti-GPC3 CSR 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 caTCR, anti-GPC3 CSR, 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 having anti-GPC3 caTCR and/or anti-GPC3 CSR aggregated on the cell surface. Aggregation can be characterized by, for example, a staining pattern with uneven thickness or signal intensity, or a discontinuous, blocky, punctate, and/or uneven distribution pattern. In some embodiments, the caTCR+CSR+c-Jun T cells express anti-GPC3 caTCR and/or anti-GPC3 CSR, wherein the amount of aggregation of the anti-GPC3 caTCR and/or anti-GPC3 CSR on the cell surface is less than about 10% (e.g., less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). In some embodiments, the 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% (e.g., less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). In some embodiments, the 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, the 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 conditions of prolonged immune activation, such as in cancer or chronic infection, where T cells become functionally abnormal. 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 prevents optimal clearance of tumor cells and infections. T cell depletion 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 profile (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 depletion 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 ability 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

Also provided herein are anti-GPC3 caTCR plus anti-GPC3 CSR plus c-Jun ("caTCR+CSR+c-Jun") compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising immune cells (e.g., T cells) 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 cells. In some embodiments, the caTCR+CSR+c-Jun immune cell composition is a pharmaceutical composition.

The composition may comprise: 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 comprise cells that are not caTCR+CSR+c-Jun immune cells.

At various points during 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.

As is understood 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 196:548), albumin, polydextrose, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan and Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954, Biochem. J. 56:265), and inorganic salts (Phan and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan and Bender, 1961, Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery ed., Butterworth, London, p. 59). In certain embodiments, DMSO can be used. Adding plasma (e.g., to a concentration of 20-25%) can enhance the protective effect of DMSO. After adding DMSO, cells can be kept at 0°C until frozen, as 1% DMSO concentration can be toxic at temperatures above 4°C.

In cryopreservation of cells, a slow, controlled cooling rate may 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 to ice should be minimal. The cooling program can be performed by using, for example, a programmable freezer or a methanol bath program. The programmable freezer allows the optimal cooling rate to be determined and facilitates standard, reproducible cooling.

In a specific 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 optimal. After at least two hours, the sample can reach a temperature of -80°C and can be placed directly in liquid nitrogen (-196°C).

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 this type of storage.

Other considerations and procedures for manipulation, cryopreservation, and long-term storage of cells can be found in the following exemplary references: U.S. Patent Nos. 4,199,022, 3,753,357, and 4,559,298; Gorin, 1986, Clinics In Haematology 15(1):19-48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186; Livesey and Linner, 1987, Nature 327:255; Linner et al., 1986, J. Histochem. Cytochem. 34(9):1 123-1 135; Simione, 1992, J. Parenter. Sci. Technol. 46(6):226-32.

After cryopreservation, the frozen cells can be thawed for use according to methods known to those of ordinary skill in the art. 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 swirling will ensure that the cell suspension is mixed as it thaws and improve heat transfer from the warm water to the ice cubes inside. Once the ice is completely melted, the vial can be immediately placed on ice.

In certain embodiments, methods to prevent cell agglutination can be used during thawing. Exemplary methods include: adding deoxyribonuclease (Spitzer et al., 1980, Cancer 45:3075-3085), low molecular weight polydextrose and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24), etc. before and/or after freezing. As is understood by those of ordinary skill, if a cryoprotectant toxic to humans is used, it should be removed prior to therapeutic use. DMSO is not severely toxic.

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).

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, 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.

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.

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.

Stabilizers refer to a broad class of excipients whose functions range from those that act as bulking agents to those that help prevent cells from adhering to the walls of a container. Typical stabilizers may include polyhydroxy sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamine, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, 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 hydroxyacetate, 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 polydextrose.

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

Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, octadecyldimethylbenzamide chloride, benzyl ammonium halides, hexahydroxyquaternary ammonium chloride, alkyl p-hydroxybenzoate (such as methyl or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, and 3-pentanol.

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 greater than 10 ¹¹ cells.

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 usually greater than 10 ⁴ cells/ml, 10 ⁷ cells/ml, or 10 ⁸ cells/ml.

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, a sizing agent, a diluent, a thickening agent, a stabilizer, a buffer and/or a preservative; and/or an aqueous medium, such as purified water, a sugar solution, a buffer solution, a physiological saline solution, a polymer solution, or ribonuclease-free water. The amount of such additives and aqueous media to be added can be appropriately selected according to the form of use of the nucleic acid composition.

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.

Formulations for intravenous administration must be sterile. This requirement is readily achieved, for example, by filtering through sterile filter membranes.

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), 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. Preferably, the carrier is isotonic. In some embodiments, the carrier can be supplemented with ingredients such as human serum albumin (HSA) or other human serum components, 5% glucose or dextrose. Other isotonic agents may also be included, including polyhydroxy sugar alcohols, including trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol or mannitol.

The pharmaceutical immune cell composition (such as the pharmaceutical T cell composition) can be administered to a cancer patient systemically (e.g., by intravenous or portal vein injection) or locally (e.g., by intratumoral injection) in a therapeutically effective amount. In some embodiments, the composition (such as a composition targeting GPC3) is 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 individual. Such results include, but are not limited to, alleviation of one or more symptoms caused by a disease (e.g., HCC), reduction of the extent of the disease (e.g., reduction of tumor size), stabilization of the disease (e.g., prevention or delay of disease worsening), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence or relapse, improvement of the disease state, disease remission (partial or complete), reduction of the dosage of one or more other drugs required to treat the disease, improvement of quality of life, restoration of weight, and/or extension of survival (e.g., overall survival or progression-free survival).

A therapeutically effective amount of a composition refers to the number of engineered immune cells (such as T cells) sufficient to achieve the desired clinical indicator. In some embodiments, the therapeutically effective amount contains more than 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ or 10 ⁹ engineered cells.

In some embodiments, the pharmaceutical composition comprises an amount of cells 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 individual. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be applicable and can be determined. The desired dose may be delivered by administering the composition as a single bolus, by administering the composition as multiple boluses, or by administering the composition as a continuous infusion.

In certain embodiments, a subject is administered a range of about 1 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 3 0 billion cells, about 40 billion cells, or a range defined 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 , about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range bounded by any two of the foregoing values), and in some cases, about 100 million cells to about 500 million cells. The number of cells in the human genome can range from about 100 million to about 100 million cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value in between these ranges.

In some embodiments, cells and compositions are administered using standard administration techniques, formulations, and/or devices. Formulations and devices for storing and administering the compositions, such as syringes and vials, are provided. Administration can be autologous or allogeneic. For example, immunoreactive cells or progenitor cells can be obtained from one individual and administered to the same individual or to a different compatible individual. Immunoreactive cells or their progeny derived from peripheral blood (e.g., from an in vivo, ex vivo, or ex vivo source) can be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the invention (e.g., a pharmaceutical composition containing genetically modified cells), it is usually formulated in a unit-dose injectable form (solution, suspension, emulsion).

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, such as the above-mentioned viral vector (e.g., lentiviral vector). The pharmaceutical composition can be used in vitro to engineer T or progenitor cells, which are then introduced into the patient. The pharmaceutical composition comprises a nucleic acid molecule or recombinant virus whose genome comprises an expression cassette of anti-GPC3 caTCR, anti-GPC3 CSR and c-Jun, and a pharmaceutically acceptable carrier, such as a buffer solution, which optionally contains other reagents, such as preservatives, stabilizers and the like.

The pharmaceutical composition may be provided as a product, such as a kit, comprising a vial (e.g., a single-dose vial) containing the biological material (cells or nucleic acid molecules or recombinant viruses) and, if necessary, instructions for use. V. Methods of Treatment

The expression constructs and/or compositions (e.g., pharmaceutical compositions) of the present invention can be administered to a subject (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 (e.g., hepatocellular carcinoma (HCC)).

Therefore, in some embodiments, the present application provides a method for treating a GPC3-positive disease in an individual in need thereof, comprising administering to the individual an effective amount of a composition (e.g., 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.

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 (e.g., a pharmaceutical composition) comprising one or more immune cells expressing an anti-GPC3 caTCR, an anti-GPC3 CSR, and a c-Jun polypeptide (e.g., 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, 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. In some embodiments, the cancer is HCC. In some embodiments, the cancer is HCC and the treatment comprises preventing the spread of the cancer, e.g., inhibiting (e.g., preventing) cancer metastasis. In some embodiments, the cancer is metastatic HCC. In some embodiments, the individual is a human.

In some embodiments, provided herein is a method for treating a GPC3-positive disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition (e.g., a pharmaceutical composition) comprising immune cells expressing one or more of the following: one or more expression constructs, wherein the expression constructs comprise expression cassettes for expressing one or more of the following: a) an anti-GPC3 caTCR comprising: i) an antigen binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 caTCR comprising: i) an antigen binding module that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; CSR, comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a synergistic immune cell signaling module capable of providing a synergistic stimulatory signal to the immune cell, wherein the ligand binding module and the synergistic 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, the stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains 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, for example, the group consisting of: 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.

In some embodiments, provided herein is a method of treating a GPC3-positive disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition (e.g., a pharmaceutical composition) comprising immune cells expressing one or more of the following: one or more expression constructs comprising expression cassettes for expressing one or more of the following: a) an anti-GPC3 caTCR comprising: i) an antigen binding module comprising: (a) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20; and (b) a _VH comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising an amino acid sequence of DDS, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at _least one TCR-associated signaling molecule; b) an anti-GPC3 CSR comprising: i) a ligand binding module, the ligand binding module comprising: (a) a VH 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; and (b) a _VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15, a LC-CDR2 comprising an amino acid sequence of YDS, and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 17; ii) a transmembrane _module ; and iii) a synergistic immune cell signaling module capable of providing a synergistic stimulatory signal to the immune cell, wherein the ligand binding module and the synergistic immune cell signaling module are not derived from the same molecule, and wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the antigen binding module of the anti-GPC3 caTCR comprises a _VH comprising an amino acid sequence of SEQ ID NO: 28 and a _VL comprising an amino acid sequence of SEQ ID NO: 29. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 30 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 31. In some embodiments, the caTCR is a heterodimer comprising a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 10 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-GPC3 caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have 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 _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-GPC3 ligand binding module of the anti-GPC3 CSR comprises an 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 synergistic 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 an amino acid sequence of SEQ ID NO: 44. In some embodiments, the anti-GPC3 CSR comprises an 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 an 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.

In some embodiments, provided herein is a method for treating a GPC3-positive disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition (e.g., a pharmaceutical composition) comprising immune cells expressing one or more of the following: one or more expression constructs comprising expression cassettes for expressing one or more of the following: a) an anti-GPC3 caTCR comprising: i) an antigen binding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; b) an anti-GPC3 caTCR comprising: i) an antigen binding module comprising a Fab that specifically binds to GPC3; and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-associated signaling molecule; CSR, comprising: i) a ligand binding module comprising a scFv capable of binding or interacting with GPC3; ii) a transmembrane module derived from CD30; and iii) a synergistic immune cell signaling module derived from CD30 capable of providing synergistic stimulatory signals to immune cells, wherein the CSR lacks a functional primary immune cell signaling domain; and c) a human c-Jun polypeptide. In some embodiments, the Fab comprises a heavy chain variable domain ( _VH ) comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain ( _VL ) comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-GPC3 CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the human c-Jun polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the cancer is selected 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.

In some embodiments, provided herein is a method for treating a GPC3-positive disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition (e.g., a pharmaceutical composition) comprising one or more immune cells expressing a multicistronic expression construct, wherein the multicistronic expression construct comprises 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 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; CSR, which comprises: 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 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 NOs: 28 and 29, and the coding sequence of SEQ ID NO: 33. In some embodiments, the coding sequences are 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 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.

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

The present invention will be more fully understood with reference to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or variations thereof will be proposed by those skilled in the art and are included in the spirit and scope of this application and the scope of the attached patent application.

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 .

Two lentiviral vectors were constructed. The first, referred to herein as "GPC3-caTCR + GPC3-CD30-CSR" or "Construct 1," contained a tri-cistronic expression cassette encoding a GPC3-specific caTCR (SEQ ID NOs: 30 and 31) and a GPC3-specific CSR comprising a co-stimulatory domain derived from CD30 (i.e., GPC3-CD30-CSR) (SEQ ID NO: 16). The second vector, referred to herein as "c-Jun + GPC3-caTCR + GPC3-CD30-CSR" or "Construct 2," contains a tetra-cistronic expression cassette encoding GPC3-specific caTCR (SEQ ID NOs: 30 and 31), 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 for the different polypeptides are linked in frame by the coding sequence for a self-cleaving peptide (F2A, T2A, or P2A, as shown in FIG. 1A ). Recombinant viruses were produced by transfecting 293T cells with vectors encoding the constructs using known lentiviral production protocols and packaging systems.

Primary human T cells were used for lentiviral transduction after one day of stimulation with CD3/CD28 beads (Dynabeads®, Invitrogen) in the presence of 100 U/mL interleukin-2 (IL-2). Concentrated lentivirus was applied to T cells in 6-well plates coated with Retronectin® (Takara) and incubated for 96 hours. Transduction efficiency was assessed by flow cytometry on day 9 using PE-conjugated anti-human F(ab')2 antibodies. Flow cytometry data were collected using BD FACSCanto II™ and analyzed using FlowJo software suite.

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). HepG2 cells expressing luciferase (HepG2-luc) or Hep3B cells were implanted subcutaneously (sc) into the right flank of NSG-MHC I/II DKO mice (depicted in Figure 1B ). When tumors reached approximately 150 mm ³ in size (20 days after tumor cell implantation), mice were injected intravenously (iv) with modified T cells.

The health effects of T cell infusion in mice were evaluated by monitoring the animals' general 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 an in vitro long-term target engagement assay

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 co-stimulatory 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 depletion, T cells expressing anti-GPC3 caTCR + anti-GPC3 CSR behaved comparably in the presence or absence of c-Jun overexpression.

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 by the CellCytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released after cell lysis. CD3+ T cells were prepared from PBMC-enriched whole blood using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies), which is depleted of cells expressing CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, and glycophorin A. Human T cells were activated and expanded using CD3/CD28 Dynabeads® (Invitrogen) according to the manufacturer's protocol. 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 normalized by caTCR expression (caTCR ⁺ ) based on human F(ab')2 staining (all effector cell samples were adjusted to the same percentage of receptor ⁺ (caTCR ⁺ ) cells among the total T cells using untransduced/mock-transduced T cells).

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

A comparison of long-term killing (as represented by the amount of remaining target cells and the number of remaining T cells) is shown in Table 3. T cells expressing construct 2 eliminated target cells via more rechallenges than T cells expressing construct 1, indicating that c-Jun promotes the long-term target cell killing ability of anti-GPC3 caTCR ⁺ anti-GPC3 CSR ⁺ T cells.

After engagement of each target cell, CD4 or CD8 single positive caTCR ⁺ T cell subsets were analyzed by flow cytometry. The data in Table 3 show that c-Jun overexpression resulted in a higher percentage of CD4 ⁺ T cells but a lower percentage of CD8 ⁺ T cells, indicating that c-Jun expression promotes preferential expansion of CD4 ⁺ T cells.

To determine whether transduction with construct 1 or construct 2 had an effect on T cell differentiation during the long-term killing assay, CD8 ⁺ receptor ⁺ cells were analyzed by flow cytometry for the expression of CCR7 and CD45RA. Naive T cells are characterized by CCR7 ⁺ and CD45RA ⁺ , while memory T cells are characterized by CCR7 ⁺ and ^CD45RA- . As shown in Table 3 , the amount of CCR7 expression and the number of memory 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 the differentiation of anti-GPC3 caTCR ⁺ anti-GPC3 CSR ⁺ memory T cells.

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

As shown in Table 3 , both groups of T cells showed similar levels of exhaustion markers. These results demonstrate that, with respect to long-term exhaustion, anti-GPC3 caTCR ⁺ anti-GPC3 CSR ⁺ T cells behaved equivalently in the presence or absence of c-Jun overexpression. Table 3. In vitro long-term target engagement results Comparison features Structure 1 Structure 2 CD3 ⁺ T cells Quite HepG2 cell elimination Higher Receptor ⁺ T cells Quite CD8 ⁺ T cells in receptor ⁺ T Higher CD4 ⁺ T cells in receptor ⁺ T Higher CCR7 expression in CD8 ⁺ receptor ⁺ T cells Quite CD8 ⁺ receptor ⁺ T in the initial T cells Quite CD8 ⁺ receptor ⁺ central memory in T Quite PD1 CD8 ⁺ receptor ⁺ Quite CD4 ⁺ receptor ⁺ Quite LAG3 CD8 ⁺ receptor ⁺ Quite CD4 ⁺ receptor ⁺ Quite TIM3 CD8 ⁺ receptor ⁺ Quite CD4 ⁺ receptor ⁺ Quite Example 2 : In vivo efficacy of anti -GPC3 caTCR + anti- GPC3 CSR T cells overexpressing c-Jun in a rechallenge study in a HepG2-luc mouse model

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.

As described above, primary human T cells from two different donors expressing construct 1 or construct 2 were evaluated for their 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). 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 approximately 70 days after T cell administration.

In all four treatment groups (i.e., high-dose construct 1, low-dose construct 1, high-dose construct 2, low-dose construct 2), tumor regression began at approximately day 10 after T cell injection; complete tumor regression was achieved at approximately 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.

Interestingly, an enhancing effect of c-Jun was observed when the animals underwent tumor re-challenge. T cells expressing construct 2 were significantly better expressed 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 ). In addition, 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

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

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). As shown in Figure 5 , 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 indistinguishable. 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 the expansion of anti-GPC3 caTCR ⁺ anti-GPC3 CSR ⁺ T cells in vivo ( Figure 4C ). Example 4 : Characterization of T cells expressing various anti -GPC3 caTCR + anti- GPC3 CSR in in vitro long-term target engagement assays

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 co-stimulatory domain derived from human CD30 (i.e., GPC3-CD30-CSR).

Primary T cells isolated from healthy human donors were transduced with one of the expression constructs described in Table 4 below. Table 4. Exemplary expression constructs comprising different combinations of anti- GPC3 moieties Structure Description Anti-GPC3 caTCR _VH / _VL (SEQ ID NO) Anti-GPC3 CSR _VH / _VL (SEQ ID NO) V _H V _L V _H V _L c-Jun+GPC3-3 caTCR+GPC3-2-CD30 CSR 28 29 26 27 c-Jun+GPC3-3 caTCR+GPC3-3-CD30 CSR 28 29 28 29 c-Jun+GPC3-3 caTCR+GPC3-1-CD30 CSR 28 29 twenty four 25 c-Jun+GPC3-2 caTCR+GPC3-2-CD30 CSR 26 27 26 27 c-Jun+GPC3-2 caTCR+GPC3-3-CD30 CSR 26 27 28 29 c-Jun+GPC3-2 caTCR+GPC3-1-CD30 CSR 26 27 twenty four 25 c-Jun+GPC3-1 caTCR+GPC3-2-CD30 CSR twenty four 25 26 27 c-Jun+GPC3-1 caTCR+GPC3-3-CD30 CSR twenty four 25 28 29 c-Jun+GPC3-1 caTCR+GPC3-1-CD30 CSR twenty four 25 twenty four 25

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

The construct c-Jun+GPC3-3 caTCR+GPC3-2-CD30 CSR (Table 4) showed the highest long-term target cell killing ability 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

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 synergistic stimulatory fragments (e.g., synergistic stimulatory fragments comprising a synergistic stimulatory domain and a transmembrane domain).

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 _VH / _VL sequence of GPC3-3 (see Sequence Listing), and each exemplary GPC3-CSR encodes the _VH / _VL sequence of GPC3-2 (see Sequence Listing). Table 5. Exemplary expression constructs containing anti- GPC3-CSRs containing different synergistic stimulatory fragments Structure Description IC domain Transmembrane (TM) domain Anti-GPC3 caTCR _VH / _VL (SEQ ID NO) Anti-GPC3 CSR _VH / _VL (SEQ ID NO) V _H V _L V _H V _L c-Jun+GPC3-3 caTCR (no CSR) N/A N/A 28 29 N/A N/A c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM-CD30 IC-CSR CD30 CD30 28 29 26 27 c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR CD30 CD28 28 29 26 27 c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD28 IC-CSR CD28 CD28 28 29 26 27 c-Jun+GPC3-3 caTCR+GPC3-2-4-IBB TM-4-IBB IC-CSR 4-IBB 4-IBB 28 29 26 27

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

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

T cells expressing each of the constructs described in Table 5 were co-cultured with HepG2 cells and tumor cell cytotoxicity was analyzed by the Cytox 96® Non-Radioactive Cytotoxicity Assay (Promega), which quantitatively measures lactate dehydrogenase (LDH) released into the culture supernatant, as described in Example 1.

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.

The long-term killing capacity of various T cells of the constructs shown in Table 5 was also determined by measuring the amount/level of interleukins released from T cells after conjugation with HepG2 cells. The level of interleukin release in the supernatant after 16 hours of co-culture was quantified using the BioRad Bio-Plex kit or by ELISA using Luminex Magpix technology. T cells with higher cytotoxic potency secrete high levels of interleukins associated with T cell activity, such as TNFα, GM-CSF, IFNγ, and IL-2.

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSRs containing at least a CD30 IC domain have higher killing efficacy than those T cells without any CSRs, and higher or about the same as those T cells containing CSRs that do not contain a CD30 IC domain but have 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

The proliferation and persistence of genetically modified T cells are critical to the success of donor-acceptor T cell transfer therapy for the treatment of 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 the dye was observed to be diluted when the T cells divided upon stimulation with tumor cells. T cell persistence was measured by counting the number of CFSE-positive cells remaining on a given day.

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 were split on the indicated days, serial dilutions of the CFSE dye were observed using flow cytometry. The total number of T cells was counted using FACS.

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSRs containing at least a CD30 IC domain proliferate to a greater extent than corresponding CAR T cells without any CSRs, and to a greater extent or about the same as corresponding CAR T cells expressing CSRs that do not contain a CD30 IC domain but have a different co-stimulatory molecule IC domain (e.g., a CD28 or 4-1BB IC domain). Example 5C : In Vivo T Cell and Tumor Cell Counts After Multiple Weeks of Engagement

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 entrainment.

T cells expressing c-Jun, an anti-GPC3 caTCR, and an anti-GPC3 CSR containing at least a CD30 IC domain persisted in multiple engagements with tumor cells for longer periods and killed more tumor cells than corresponding CAR T cells without the CSR, and were about the same (if not better) than CAR T cells containing a CSR without the CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain).

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. The number of remaining target cells and total T cells was quantified by FACS at different days after each target cell conjugation.

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSRs containing 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 counterpart T cells without any CSRs, and survive better and/or kill more tumor cells than or about the same as counterpart T cells expressing CSRs without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5D : In Vitro T Cell Depletion

To examine the levels of exhaustion markers expressed on T cells transduced with the constructs described in Table 5, the expression of exhaustion markers TIGIT, PD-1, LAG-3, and TIM-3 (BioLegend®) on caTCR ⁺ CD8 ⁺ T cells and caTCR ⁺ CD4 ⁺ T cells was analyzed by flow cytometry on selected days after engagement of each target cell, as described in Example 1.

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR containing at least a CD30 IC domain have reduced long-term exhaustion markers compared to corresponding T cells without any CSR or T cells expressing CSR without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5E : Interleukin release in vivo

Primary human T cells expressing each of the constructs in Table 5 were evaluated for in vivo cytotoxic potency in the HepG2-luc and Hep3B mouse models as described in Examples 2 and 3, respectively. Briefly, to determine in vivo interleukin release levels, key interleukins, including those associated with clinical interleukin release syndrome, were analyzed 16, 24, 48, and 72 hours after administration of T cells. Interleukin levels were quantified by Luminex Magpix technology using the BioRad Bio-Plex kit.

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR containing at least a CD30 IC domain secrete higher levels of interleukins associated with T cell activity, such as TNFα, GM-CSF, IFNγ, and IL-2, than counterpart T cells without any CSR or expressing CSR without a 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

In two independent flow cytometry assays, proliferation and survival of T cells expressing the expression constructs described in Table 5 were measured before and after target cell engagement.

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 and naive T cells compared to T cells without any CSR or T cells expressing CSR without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain).

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3-CD30-CSR developed and maintained high populations of memory T cells, including central memory T cells and effector memory T cells, after targeted stimulation. To determine the effects of different CSRs on the ability of T cells to develop and maintain memory T cells, cell surface expression of the memory T cell markers CCR7 and CD45RA was measured. As is known in the art, T cells with high CCR7 expression and low CD45RA expression are considered central memory T cells, T cells with low CCR7 and low CD45RA expression are considered effector memory T cells, T cells with low CCR7 and high CD45RA expression are considered effector T cells, and T cells with high CCR7 and high CD45RA are naive T cells, i.e., the initial type of T cell prior to target/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 work to destroy the target and subsequently die, while a small pool of T cells eventually develop into long-term memory T cells that can store T cell immunity against a specific target. Among memory T cells, central memory T cells were found to have a longer lifespan than effector memory T cells and are capable of generating effector memory T cells, but not vice versa. Therefore, the ability to develop and maintain memory T cells, especially central memory T cells, is an important and desired feature for a potentially successful T cell therapy.

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

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

On selected days after each HepG2 cell junction, the entire cell mixture from each sample well was stained with antibodies against CCR7 and CD45RA and analyzed by flow cytometry. The number of receptor ⁺ T cells was counted and the cells were grouped into various T cell types based on the amount of CCR7 and CD45RA expression on the cells: central memory T cells (CD45RA ^- CCR7 ⁺ ), effector memory T cells (CD45RA ^{- CCR7-} ), effector T cells (CD45RA+CCR7- ⁾ , and primary T cells (CD45RA ⁺ CCR7 ⁺ ). The percentage of each type of T cell among the total number of receptor ⁺ T cells was calculated. 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.

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 containing at least a CD30 IC domain were able to develop into and maintain high numbers and percentages of central memory T cells after engagement with HepG2 cells, compared to T cells without any CSR or T cells expressing CSRs without a CD30 IC domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or 4-1BB IC domain). Example 5G : In vivo efficacy of T cells in the HepG2 or Hep3B mouse model

Approximately 10 ⁷ HepG2 tumor cells or Hep3B were implanted subcutaneously in NSG mice and allowed to form 150 mm ³ solid tumor masses. 5×10 ⁶ T cells representing each of the constructs described in Table 5 were injected intravenously into tumor-bearing mice. Three weeks after T cell administration, mice were sacrificed and tumors were removed, fixed and sectioned onto slides. Tumor sections were stained with CD3 antibodies to visualize T cells present within solid tumors. Quantification of CD3 ⁺ cell numbers can be used to score the tumor infiltration capacity of T cells (T cells/mm ² ).

T cells expressing c-Jun, anti-GPC3 caTCR, and anti-GPC3 CSR comprising at least a CD30 IC domain have higher in vivo tumor infiltration/penetration rate/level (i.e., higher number of T cells/mm 2 ) than T cells without any CSR or T cells expressing CSR without a CD30 domain but with a different co-stimulatory molecule IC domain (e.g., CD28 or ^4-1BB IC domain). SEQUENCE LISTING SEQ describe sequence 1 Human c-Jun MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF 2 Signal peptide METDTLLLWVLLLWVPGSTG 3 Partial TCR γ QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 4 Partial TCR δ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 5 caTCR-2 peptide 2 QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 6 GPC3-1 HC-CDR1 GFTFSSYA 7 GPC3-1 HC-CDR2 YYG 8 GPC3-1 HC-CDR3 ARTSYLNHGDY 9 GPC3-1 LC-CDR1 RSNIGSDY 10 caTCR-2 peptide 1 QVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSSQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 11 GPC3-1 LC-CDR3 GTWDYTLNGVV 12 GPC3-2 HC-CDR1 GYTFTSYY 13 GPC3-2 HC-CDR2 INPSGGST 14 GPC3-2 HC-CDR3 ARWHGGPYDY 15 GPC3-2 LC-CDR1 NIGSKS 16 CSR - 1 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSAAATGAPPLGTQPD CNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 17 GPC3-2 LC-CDR3 QVWDSSSDHYV 18 GPC3-3 HC-CDR1 GYTFASHG 19 GPC3-3 HC-CDR2 ISPYTGNT 20 GPC3-3 HC-CDR3 ARGKRTLASCFDY twenty one GPC3-3 LC-CDR1 NIGSKS twenty two CD8 transmembrane domain TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC twenty three GPC3-3 LC-CDR3 QVWDSSSDHV twenty four GPC3-1 V _H QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSS 25 GPC3-1 V _L QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLG 26 GPC3-2 V _H QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSS 27 GPC3-2 V _L QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLG 28 GPC3-3 V _H QVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSS 29 GPC3-3 V _L QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLG 30 caTCR-1 peptide1 QVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 31 caTCR-1 peptide2 QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 32 GPC3-1 scFv QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSS 33 GPC3-2 scFv QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSS 34 GPC3-3 scFv QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSS 35 TCRα homeostasis domain PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 36 TCRβ homeostasis domain EDLCLVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 37 TCRδ constant domain SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 38 TCRγ constant domain DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 39 TCRα transmembrane domain ILLLKVAGFNLLMTLRLWSS 40 TCRβ transmembrane domain TILYEILLGKATLYAVLVSALVL 41 TCRδ transmembrane domain VLGLRMLFAKTVAVNFLLTAKLFFL 42 TCRγ transmembrane domain (same as uniprot) YYMYLLLLLKSVVYFAIITCCLL 43 CD30 synergistic stimulatory fragment 1 APPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 44 CD30 synergistic stimulatory fragment 2 HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 45 CD28 synergistic stimulatory fragment 1 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 46 CD28 synergistic stimulatory fragment 2 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 47 4-1BB Synergistic Stimulation Clip 1 PADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 48 4-1BB Synergistic Stimulation Clip 2 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 49 CD30 transmembrane domain PVLDAGPVLFWVILVLVVVVGSSAFLLC 50 CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV 51 CD8 transmembrane domain IYIWAPLAGTCGVLLLSLVIT 52 c-Jun Nucleotide Sequence #1 atgacagccaagatggaaaccacattctacgacgacgccctgaacgcctcattcctgccttctgagagcggaccttacggctacagcaatcctaagatcctgaaacagagcatgacccttaacctggctgatcctgttggaagcctgaaacctcacctgagagccaaaaacagcgacctgctcaccagccctgatgtgggcctgctgaagctggcctctccagagctggaacggctgatcatccagag cagcaacggccacatcacaaccacccctacccctacacaattcctgtgccctaagaacgtgaccgacgagcaggagggcttcgccgaaggctttgtgcgggccctggcagaactgcactctcagaacaccctgcctagcgtgacctccgccgcccagcctgtcaacggcgccggaatggtggcccctgccgtggcttctgtggccggcggcagcggcagcggcggattcagcgcctctctgcactctg agcctcctgtctacgccaatctgtctaatttcaaccccggagccctgtccagcggcggcggagctcctagctacggcgctgctggactggccttccccgcccagccccagcaacagcagcctccacaccacctgccccagcagatgcccgtgcagcaccctagactgcaggccctgaaggaagaaccccaaacagtgcctgagatgcctggcgagacacctccactgagccccatcgacatggaa agccaggagcggatcaaggccgagagaaagagaatgcggaacagaatcgccgctagcaagtgcagaaagcggaagctggaaagaatcgccagactggaagagaaggtgaagaccctgaaagcccaaaatagcgagctggccagcaccgccaacatgctgcgggaacaggtggcccagctgaagcagaaggtgatgaaccacgtgaactctggttgtcagctgatgctgacccagcagctccagaccttc 53 c-Jun Nucleotide Sequence #2 atgacagccaagatggaaaccaccttctacgacgacgccctcaacgcctccttcctgccttctgagagcggtccttacggctacagcaaccccaagatcctgaagcaaagcatgaccctgaacctggccgaccccgttggctccctgaaacctcacctgagagccaaaaacagcgacctgctgaccagccctgatgtgggcctgctgaagctggcctctccagagctggaaagactgattatccagag cagcaacggccacatcaccacaacacctacccctacacagttcctgtgccctaagaacgtgactgatgagcaggagggctttgccgagggcttcgtgagagccctggctgagctgcattctcagaacaccctgcctagcgtgacctctgccgcccagcctgttaatggcgccggcatggtggcccctgccgtggcctctgtggccggaggcagcggcagcggcggattcagcgcctctctgcacagcg agccccccgtctacgccaacctgagcaatttcaaccctggcgccctgtccagcggcggcggcgccccttcatatggcgctgccggcctggccttccccgctcagccccagcagcagcaacagcctccacaccacctgccccagcagatgcccgtgcagcaccccagactgcaggccctgaaggaagaacctcagaccgtgcccgagatgcctggcgagacccctcctctgagccctatcgacatggaa agccaggagagaatcaaggccgagaggaagcggatgcggaacagaatcgccgccagcaagtgcagaaaaagaaagctggaacggatcgccagactggaggagaaggtgaagacactgaaagcccaaaattctgaactggcctctaccgccaatatgctgcgcgagcaggtggctcaactgaagcagaaggtgatgaaccacgtgaacagcggatgtcagctgatgctgacacagcagctgcagactttt 54 c-Jun Nucleotide Sequence #3 atgaccgccaagatggaaaccaccttctacgacgacgccctgaacgccagctttctgccttctgagtctggcccctacggctacagcaaccccaagatcctgaagcagagcatgaccctgaacctggccgatcctgtgggcagcctgaaacctcacctgagagccaagaacagcgacctgctgacaagccctgatgtgggcctgctgaaactggcctctcctgagctggaacggctgatcatccagag cagcaacggccacatcaccaccacacctacaccaacacagtttctgtgccccaagaacgtgaccgacgagcaagagggattcgccgagggctttgttagagccctggccgaactgcacagccagaataccctgcctagcgtgacatctgccgctcagcctgttaatggcgccggaatggttgctcctgccgtggcttctgttgctggcggatctggatctggcggctttagcgcctctctgcactctg agcctccagtgtacgccaacctgagcaacttcaaccctggcgctcttagctctggtggcggagcaccttcttatggcgctgccggattggcctttcctgctcagcctcagcagcagcaacagcctcctcatcatctgccccagcagatgcctgtgcagcaccctagactgcaggccctgaaagaggaaccccagacagtccctgagatgcccggcgaaacacctcctctgagccccatcgacatggaa agccaagagcggatcaaggccgagcggaagcggatgagaaatagaatcgccgcctccaagtgccggaagaggaagctggaaagaatcgcccggctggaagagaaagtgaaaaccctgaaggcccagaactccgagctggcctctaccgccaacatgctgagagaacaggtggcccagctgaaacagaaagtcatgaaccacgtgaacagcggctgccagctgatgctgacacagcagctgcagaccttc 55 c-Jun Nucleotide Sequence #4 atgactgccaaaatggagactacattctatgacgacgccctcaatgccagttttttgccgagtgaatccggcccctacggctattcaaaccctaagatcctcaagcaatcaatgaccctcaatcttgctgacccagttggctccctgaaaccccatctcagagctaaaaatagtgacctccttacttcccctgatgttggactcctcaaacttgcttctcccgaactcgaacgcttgatcattcaatc ttccaacggccacatcacaacaacacccacacccacccagtttctttgcccaaaaaatgtcaccgatgaacaggaaggtttcgcggaaggattcgtccgcgcgctggccgaactgcactcccagaatacacttccttcagttacgtcagccgcccagccagtgaatggtgcgggaatggttgctcctgcggtcgcttctgtcgcagggggctccggttctggcggatttagcgcctctctgcattccg agccacctgtatatgctaatctttctaattttaaccccggagccttgtctagcggcggtggtgcccccagctacggtgctgcaggactcgccttcccagctcaacctcagcagcaacaacccccccatcaccttccccaacagatgccagtacaacatccaaggctccaggccctcaaagaggaaccacagacggtgcccgaaatgcctggcgaaactccaccactttcccctattgatatggaa tcccaagagcgcatcaaggccgaaagaaagcgaatgcggaatagaatagcagcttcaaaatgtagaaaacggaaattggaacgaatcgcacggttggaagaaaaggtgaagaccttgaaagcccagaacagtgagctcgcctctaccgctaacatgctgcgcgagcaagtcgcacaacttaagcagaaggtgatgaaccatgtgaatagcggatgtcaacttatgctgactcaacagttgcaaaccttt 56 c-Jun Nucleotide Sequence #5 atgaccgcgaaaatggagacaacattttacgatgatgcactgaacgcctcttttctgccaagtgaatccggcccctacggatactcaaaccctaagattctgaaacagtctatgactctcaacctggccgacccagttggcagtctgaagcctcatttgcgagccaagaatagtgatctgctgacctccccagacgtgggactgctgaaactcgcctcacctgaacttgagcgcttgattatacagtc atccaatgggcacatcacaacaacacctactcctacccagtttctgtgccccaaaaacgtcaccgatgagcaggagggattcgcggaaggctttgtgcgcgccctggctgaattgcatagtcagaacactcttcccagcgtaaccagcgccgcccaaccagtgaatggagccggtatggtggctcccgcggtggctagtgttgcgggggggtcaggctctggtgggttcagtgcttctcttcactctg aaccccctgtgtatgccaatctgtctaactttaaccctggggccctctcctctggtgggggtgcccccagctacggagcggccggcctggcctttcctgcccagcctcagcagcagcaaccccctcatcatcttccgcagcagatgccagtacagcatccacgcctgcaggctcttaaggaggagccccagacggtgcccgaaatgcccggggaaactccacccttgtcccccattgacatggag tcccaggagcggatcaaggctgaaagaaagaggatgcggaatcgcatcgcagcctctaaatgccgcaagcggaaacttgagaggatcgcgcggttggaggaaaaagtaaaaaccttgaaggcacagaactctgagctggcgagtactgccaacatgctcagagaacaagtcgcacagctgaagcagaaagtgatgaaccatgtgaacagcggttgtcagctgatgctgactcagcagctgcagaccttc 57 c-Jun Nucleotide Sequence #6 atgaccgccaagatggagaccacattctacgatgacgctctgaacgcttcctttctgccttccgagtccggcccctacggctactccaatcccaagattctgaagcagagcatgacactgaatctggctgatcccgtgggatctctgaagcctcatctgagagccaagaattccgatctgctgacaagccccgacgtgggactgctcaaactggccagccccgaactggagaggctcattatccagag ctccaacggccacatcaccacaacacctacccctacccagtttctctgtcccaagaacgtgacagacgagcaagagggatttgccgaaggcttcgtgagagccctcgccgaactgcatagccagaacacactgccttccgtgaccagcgctgctcaacccgtgaacggcgctggcatggtcgctcccgccgtcgccagcgtggctggaggaagcggatccggaggcttcagcgcttccctccacagcg aacctcccgtgtacgctaatctgagcaacttcaaccccggcgctctgagcagcggaggaggagctcctagctatggagctgccggactggcttttcccgcccagccccagcagcagcagcccccccatcatctgcctcagcagatgcccgtgcagcatcccagactccaagctctgaaggaggagcctcagaccgtccccgagatgcccggcgaaaccccccctctgtcccccatcgacatggaa agccaagagaggatcaaggccgagaggaagaggatgaggaatagaatcgccgccagcaagtgtagaaagaggaagctggagaggatcgccagactggaggagaaggtgaagaccctcaaggctcagaattccgagctggccagcacagccaacatgctgagagagcaagtggcccagctcaagcagaaggtgatgaaccacgtcaacagcggatgccagctgatgctcacccagcagctgcagaccttc 58 c-Jun Nucleotide Sequence #7 atgaccgctaaaatggaaaccactttctatgacgatgccctgaacgcctccttccttccgtccgagtccggaccctacggatactcaaatcctaagatcctcaaacagtcgatgaccctcaacctggccgaccccgtgggatccctgaagccgcacttgcgcgccaagaactccgacctcctgacgagcccagacgtgggcctgctgaagctcgcatcacccgaacttgagcggttgatcattcagtc ctccaacggacatatcaccaccactcccaccccaactcagtttctgtgtccgaagaacgtgaccgatgagcaagagggattcgccgagggattcgtgcgggccctggccgagctgcatagccagaacacccttccatccgtgacctcggcggctcagcctgtgaacggcgcgggaatggtcgcgcccgccgtggcctcggtggccgggggcagcggcagcgggggattttccgcgtcgctgcactccg agccgccggtgtacgccaacctgtcaaacttcaaccctggggccctgagctccggcggtggagcaccttcgtacggcgccgctggcctggcgttccccgcgcaaccacagcagcaacagcagccccctcaccacctcccccaacaaatgcctgtgcagcacccgaggctgcaggccctcaaggaagaaccccagactgtgccggaaatgccgggggagactccgccgctgtcccctatcgacatggaa tcacaggaacgcattaaggcagagcggaagcgcatgcggaaccggattgccgcctccaagtgccgcaagagaaagctcgaaagaatcgccagattggaagaaaaggtcaagactctgaaggcccagaactctgagctggcatccaccgctaatatgctgagggaacaagtggcccagctgaaacagaaggtcatgaaccacgtcaacagcggttgccagctgatgctgacccagcaactccagacattc 59 c-Jun Nucleotide Sequence #8 atgaccgccaagatggagaccaccttctacgacgacgccctgaacgccagcttcctgcccagcgagagcggaccctacggctactctaaccccaagatcctgaaacagagcatgacactgaatctggccgaccccgtgggcagcctgaagcctcaccttagagccaagaacagcgacctgctgaccagccccgacgtgggcctgctgaagctcgcctctccagagttagagagactgatcatccagtc cagcaacggccacatcacaaccaccccaacccctacccagttcctgtgccccaagaacgtgaccgacgagcaggagggcttcgccgagggctttgtgagagccctggccgagttgcactctcagaacaccctgccctccgtgaccagcgccgctcaacctgtgaacggcgcaggaatggttgctcctgccgtggccagcgttgcaggcggatctggaagtggaggcttctccgcctcccttcacagcg agcctcccgtgtacgccaacctgagcaacttcaaccccggcgccctgagcagtggaggaggcgctcccagctatggagcagctggattagccttccccgcccagccacagcagcaacagcctccccaccacctgcctcagcaaatgcctgtgcagcaccctcggctgcaggcccttaaggaggagccccagaccgttcctgagatgcctggcgagacccctcccctgagccctatcgacatggag tcccaggagcggatcaaggccgagcggaagcggatgcggaaccggatcgctgcttccaagtgccggaagagaaagctggagagaatcgcccggctggaggagaaggtgaagaccctgaaggcccagaactccgagctggcctccaccgccaacatgctgcgggagcaggttgcacagctgaagcagaaggtcatgaaccacgtgaacagcggctgccagctgatgctgacccagcagctgcagaccttc 60 c-Jun Nucleotide Sequence #9 atgacagcgaagatggagacaaccttctatgacgatgctcttaacgcctccttcctgccttccgaaagcgggccctacgggtactctaatcctaagatacttaagcaatcgatgactctcaacctcgctgacccggttggctcactgaaaccacacctgagagctaagaatagtgacctgctcactagtcccgatgtcgggcttctgaagctggcctctcccgagctggagaggcttatcatccaatcatcaaatggccacatc accactaccccaacaccaactcaattcctttgccctaaaaacgtgaccgacgaacaggaaggcttcgccgagggttttgtccgggccttggccgagctgcattctcaaaatacactgccaagcgtcacttctgcggcgcagccggttaacggagcagggatggtggctcccgccgttgctagcgtggccggcggttccggctccggcggtttctctgcctccttgcattctgagccaccagtctacgcgaacctgtccaacttta atccgggggcgctgagtagcggaggcggcgcccctagctatggggcagctggactggccttcccggcacaaccccaacaacaacagcaaccgccacaccatcttcctcaacaaatgccagtgcaacatccacgcttacaagccctcaaggaggaaccccagaccgtgcctgagatgcccggcgaaaccccgccattgagccctattgacatggaaagtcaagagagaattaaggcagagcgcaagagaatgaggaaccggatcgc agcatctaagtgccgcaaacggaaattggagcggatcgctcgcttggaggagaaggtcaagactctcaaggcccagaactccgagcttgcgagcacagctaatatgctgcgcgagcaggtggcccagttaaaacaaaaggtcatgaaccatgtgaacagcggctgtcagctgatgcttacgcaacagctgcaaacctttggctccggtgcaacgaacttcagcctgctgaagcaggccggagatgttgaggaaaatccaggtccc 61 c-Jun Nucleotide Sequence #10 atgacggccaaaatggagactacgttctacgatgacgcactcaacgcgtccttcctgccctctgagagtggaccctatggctactccaatccaaagatcctgaagcagtctatgaccctcaacctggcggacccggtgggctcccttaagccgcacttgcgcgccaagaactccgacctgctgacctcccctgatgtgggcctcctcaagctcgctagccctgaattggagaggctgatcatccagag ctcaaatggccacatcaccaccacacctaccccaacccagttcctgtgcccaaaaaacgtgaccgacgagcaggagggcttcgcggagggcttcgtcagagctctggccgagctgcactcacagaacacgctcccttccgtgacctccgctgcccagccggtcaatggcgctggaatggtggctccggctgtggcctctgttgccggcggctccggctccggaggcttttcagcttctctgcattctg agcccccagtgtacgctaacctgagcaacttcaaccccggggcgctcagctccggtggcggtgccccgagctacggcgcggctgggctggcgttccccgctcagcctcagcagcaacagcaacctccccaccacctgccacagcagatgcctgtgcagcacccacgcctgcaggccttgaaggaggaacctcagactgtgccagagatgcccggcgagaccccacccctgtccccgattgacatggag agccaggagcgcatcaaggcagagcgcaagcgtatgcgcaaccgcatcgcggcctccaagtgccgaaagcgcaagctggagcggattgctcgcctggaggagaaggtgaagaccctgaaggcccagaattccgagctggcctcgaccgccaacatgctacgagaacaggtcgcgcagctgaaacagaaggtcatgaaccatgtcaacagcgggtgccagctgatgttgacccagcagcttcagaccttc 62 Wild-type Jun nucleotide sequence (GenBank accession number NM_002228.4) *The coding region is capitalized gctcagagttgcactgagtgtggctgaagcagcgaggcgggagtggaggtgcgcggagtcaggcagacagacagacacagccagccagccagtcggcagtatagtccgaactgcaaatcttattttcttttcaccttctctctaactgcccagagctagcgcctgtggctcccgggctggtgtttcgggagtgtccagagagcctggtctccagccgcccccgggaggagagccctgctgcccaggcgctgttgacagcggcggaaagcagcggtacccacgcgcccgccgggggaagtcggcgagcggctgcagcagcaaagaactttcccggctgggaggaccggagacaagtggcagagtcccggagccaacttttgcaagcctttcctgcgtcttaggcttc tccacggcggtaaagaccagaaggcggcggagagccacgcaagagaagaaggacgtgcgctcagcttcgctcgcaccggttgttgaacttgggcgagcgcgagccgcggctgccgggcgccccctccccctagcagcggaggaggggacaagtcgtcggagtccgggcggccaagacccgccgccggccggccactgcagggtccgcactgatccgctccgcggggagagccgctgctctgggaagtgagttcgcctgcggactccgaggaaccgctgcgcacgaagagcgctcagtgagtgaccgcgacttttcaaagccgggtagcgcgcgcgcgagtcgacaagtaagagtgcgggaggcatcttaattaaccctgcgctccctggagcgagctggtgaggagggc gcagcggggacgacagccagcgggtgcgtgcgctcttagagaaactttccctgtcaaaggctccggggggcgcgggtgtcccccgcttgccacagccctgttgcggccccgaaacttgtgcgcgcagcccaaactaacctcacgtgaagtgacggactgttctATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCGGACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGGGAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAGCTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCC AGTCCAGCAACGGGCACATCACCACCACGCCGACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGCTTCGCCGAGGGCTTCGTGCGCGCCCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCAGGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGCGCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCACCTGCCCCAGCAGATGCCCGTG CAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCGAGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAGGAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCAGTTGCAAACATTTtgaagagagaccgtcgggggctgaggggcaacgaagaaaaaaaataacacagagagacagacttg agaacttgacaagttgcgacggagagaaaaaagaagtgtccgagaactaaagccaagggtatccaagttggactgggttgcgtcctgacggcgcccccagtgtgcacgagtgggaaggacttggcgcgccctcccttggcgtggagccagggagcggccgcctgcgggctgccccgctttgcggacgggctgtccccgcgcgaacggaacgttggacttttcgttaacattgaccaagaactgcatggacctaacattcgatctcattcagtattaaaggggggagggggagggggttacaaactgcaatagagactgtagattgcttctgtagtactccttaagaacacaaagcggggggagggttggggaggggcggcaggagggaggtttgtgagagcgaggct gagcctacagatgaactctttctggcctgccttcgttaactgtgtatgtacatatatatattttttaatttgatgaaagctgattactgtcaataaacagcttcatgcctttgtaagttatttcttgtttgtttgtttgggtatcctgcccagtgttgtttgtaaataagagatttggagcactctgagtttaccatttgtaataaagtatataatttttttatgttttgtttctgaaaattccagaaaggatatttaagaaaatacaataaactattggaaagtactcccctaacctctttttctgcatcatctgtagatactagctatctaggtggagttgaaagagttaagaatgtcgattaaaatcactctcagtgcttcttactattaagcagtaaaaactgt tctctattagactttagaaataaatgtacctgatgtacctgatgctatggtcaggttatactcctcctcccccagctatctatatggaattgcttaccaaaggatagtgcgatgtttcaggaggctggaggaaggggggttgcagtggagagggacagcccactgagaagtcaaacatttcaaagtttggattgtatcaagtggcatgtgctgtgaccatttataatgttagtagaaattttacaataggtgcttattctcaaagcaggaattggtggcagattttacaaaagatgtatccttccaatttggaatcttctctttgacaattcctagataaaaagatggcctttgcttatgaatatttataacagcattcttgtcacaataaatgtattcaaataccaa 63 caTCR-3 peptide 1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 64 caTCR-3 peptide 2 QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 65 caTCR-4 peptide 1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 66 caTCR-4 peptide 2 QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 67 caTCR-5 peptide 1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 68 caTCR-5 peptide 2 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 69 caTCR-6 peptide 1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 70 caTCR-6 peptide 2 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS

The drawings illustrate certain embodiments having the features and advantages of the present invention. These embodiments are not intended to limit the scope of the appended patent claims in any way.

Figure 1A is a 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 anti-GPC3 caTCR ("anti-GPC3-caTCR") plus anti-GPC3 CSR containing a co-stimulatory domain derived from CD30 ("anti-GPC3-CD30-CSR"), while construct 2 encodes "anti-GPC3-caTCR" + "anti-GPC3-CD30-CSR" and "c-Jun" polypeptide.

FIG. 1B is a schematic representation of the design of in vivo efficacy studies of T cells expressing anti-GPC3-caTCR and anti-GPC3-CD30-CSR in two different mouse models in the presence or absence of c-Jun overexpression.

FIG2 is a graph showing the in vivo tumoricidal 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. T cells were isolated from two healthy human donors. For each T cell type, two doses (5×10 ⁶ and 2×10 ⁶ ) were tested.

FIG3 is a graph showing the in vivo tumoricidal 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 rechallenge studies, in which animals were re-implanted with tumor cells after initial tumor regression.

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

FIG. 5 is a graph showing the in vivo tumoricidal 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.

TW202413646A_112126959_SEQL.xml

Claims (83)

One or more expression constructs comprising one or more expression cassettes for expressing: a) a chimeric antibody T cell receptor (TCR) construct (caTCR) comprising: i) an antigen binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM promotes the recruitment of at least one TCR-related signaling molecule; b) a chimeric stimulatory receptor (CSR) comprising: i) a ligand binding module capable of binding or interacting with GPC3; ii) a transmembrane module; and iii) a synergistic immune cell signaling module capable of providing a synergistic stimulatory signal to the immune cell, wherein the ligand binding module and the synergistic 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. The expression construct of claim 1, wherein the c-Jun is wild-type human c-Jun. 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. The expression construct of claim 1, wherein the c-Jun is a mutant human c-Jun. The expression construct of claim 4, wherein the mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain. 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. The expression construct of any one of claims 1 to 6, wherein the GPC3 is cell surface-bound GPC3. The expression construct of any one of claims 1 to 7, wherein the TCRM is derived from a human γ/δ TCR. An expression construct as in any one of claims 1 to 8, wherein the caTCR further comprises a stabilization module, the 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. The expression construct of claim 9, wherein the stabilization module is selected from the group consisting of a TCR constant domain, a C _H 1-C _L module, a C _H 2-C _H 2 module, a C _H 3-C _H 3 module, and a C _H 4-C _H 4 module. The expression construct of claim 10, wherein the stabilization module is derived from a human protein. The expression construct of claim 10 or 11, wherein the _CL contained in the _CH1 - _CL module is derived from a kappa antibody light chain or a lambda antibody light chain. The expression construct of any one of claims 1 to 12, wherein the antigen binding module of the caTCR is Fab, Fab', (Fab') ₂ , Fv or single-chain Fv (scFv). The expression construct of any one of claims 1 to 13, wherein the antigen binding module of the caTCR comprises: (i) a heavy chain variable domain (VH) 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 ; and a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 9 (ii) a light chain variable domain (VL) comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising at most about 3 amino acid substitutions; and _{a VH} comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14 or _a variant thereof comprising at most about 3 amino acid substitutions; 15 or a variant thereof comprising at most about 3 amino acid substitutions, a LC-CDR2 comprising the amino acid sequence of YDS or a variant thereof comprising at most about 3 amino acid substitutions, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising at most about 3 amino acid substitutions; or (iii) a _VL comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising at most about 3 amino acid substitutions; and a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: ₁₈ or a variant thereof comprising at most about 3 amino acid substitutions, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising at most about 3 amino acid substitutions, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising at most about 3 amino acid substitutions; 21 or a variant thereof comprising at most about 3 amino acid substitutions, LC-CDR2 comprising the amino acid sequence of DDS or a variant thereof comprising at most about 3 amino acid substitutions, and _VL comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising at most about 3 amino acid substitutions. The expression construct of claim 14, wherein the antigen binding module of the caTCR comprises: (i) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 8; and a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising an amino acid sequence of GDN, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11; (ii) a _{VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 14; and a VL comprising} an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9, an LC-CDR2 comprising an amino acid sequence of GDN, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11. or (iii) a _{VH 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; and a VL 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. The expression construct of any one of claims 1 to 15, wherein the antigen binding module of the caTCR comprises: (i) a _VH comprising an 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, and a _VL comprising an 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; (ii) a VH comprising an 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, and a _VL comprising an 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; or (iii) a _VH comprising an 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, and a _VL comprising an amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 29 or a V _L of an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 29. The expression construct of claim 16, wherein the antigen binding module of the caTCR comprises: (i) a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; or (iii) a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29. The 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. The expression construct of any one of claims 1 to 18, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising _VH fused to a TCRD derived from a TCR delta chain and a second polypeptide chain comprising _VL fused to a TCRD derived from a TCR gamma chain. The expression construct of any one of claims 1 to 18, wherein the caTCR is a heterodimer comprising a first polypeptide chain comprising _VH fused to a TCRD derived from a TCR gamma chain and a second polypeptide chain comprising _VL fused to a TCRD derived from a TCR delta chain. An expression construct as in any one of claims 1 to 19, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. An expression construct as in any one of claims 1 to 18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. An expression construct as in any one of claims 1 to 19, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. An expression construct as in any one of claims 1 to 18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. An expression construct as in any one of claims 1 to 19, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. An expression construct as in any one of claims 1 to 18 and 20, wherein the caTCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the 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 the 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. 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. 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. An expression construct as in 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. 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. The expression construct of any one of claims 1 to 30, wherein the CSR co-stimulatory immune cell signaling module is derived from human CD30. The expression construct of claim 31, wherein the human CD30 synergistic immune cell signaling module comprises the amino acid sequence of SEQ ID NO: 44. An expression construct as in any one of claims 1 to 32, wherein the synergistic immune cell signaling module and the transmembrane domain of the CSR are derived from human CD30. The expression construct of any one of claims 1 to 33, wherein the ligand binding module of the CSR comprises: (i) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 3 amino acid substitutions, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to about 3 amino acid substitutions, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 8; and a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 amino acid substitutions, an LC-CDR2 comprising an amino acid sequence of GDN or a variant thereof comprising up to about 3 amino acid substitutions, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 3 amino acid substitutions; (ii) a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 amino acid substitutions, : (i) a VH comprising a HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 3 amino acid substitutions, a HC-CDR2 comprising an 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 an amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 3 amino acid substitutions; and a _VL comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising up to about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of YDS or a variant thereof comprising up to about 3 amino acid substitutions, and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising up to about 3 amino acid substitutions; or (iii) comprising a _VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 15 or a variant thereof comprising up to about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of YDS or a variant thereof comprising up to about 3 amino acid substitutions, and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 17 or a variant thereof comprising up to about 3 amino acid substitutions; and VH comprising a LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising at most about 3 amino acid substitutions, a LC-CDR2 comprising an amino acid sequence of _DDS or a variant thereof comprising at most about 3 amino acid substitutions, and a LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23 or a _variant thereof comprising at most about 3 amino acid substitutions. The expression construct of claim 34, wherein the ligand binding module of the CSR comprises: (i) a VH 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; and a _VL 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; (ii) a _VH 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; and a _VL 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. or (iii) a _{VH 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; and a VL 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. The expression construct of any one of claims 1 to 35, wherein the ligand binding module of the CSR comprises: (i) a _VH comprising an 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, and a _VL comprising an 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; (ii) a VH comprising an 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, and a _VL comprising an 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; or (iii) a _VH comprising an 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, and a _VL comprising SEQ ID NO: The amino acid sequence of SEQ ID NO: 29 or a V _L of an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 29. The expression construct of claim 36, wherein the ligand binding module of the CSR comprises: (i) a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; (ii) a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; or (iii) a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29. 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 with SEQ ID NO: 32; (ii) SEQ ID NO: 33 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 33; or (iii) SEQ ID NO: 34 or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 34. The expression construct of any one of claims 1 to 38, wherein: (i) the antigen binding module of the caTCR comprises: (a) a VH comprising an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 18, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 19, and an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 20; and (b) a _VL comprising an LC-CDR1 comprising an amino acid sequence of SEQ ID _NO : 21, an LC-CDR2 comprising an amino acid sequence of DDS, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 23; and (ii) the ligand binding module of the CSR comprises: (a) an HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 12, an HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising an amino acid sequence of SEQ ID NO: and (b) a _VL 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 . The expression construct of any one of claims 1 to 39, wherein the antigen binding module of the caTCR comprises a Fab that binds to GPC3, wherein the ligand binding module of the CSR comprises a scFv that binds to GPC3, and wherein the transmembrane module and the co-stimulatory immune cell signaling module of the CSR are both derived from CD30. 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 _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 26 and a _VL comprising the amino acid sequence of SEQ ID NO: 27; (2) (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; (3) (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; _H and V _L comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises V _H comprising the amino acid sequence of SEQ ID NO: 24 and V _L comprising the amino acid sequence of SEQ ID NO: 25; (4) (i) the Fab comprises V _H comprising the amino acid sequence of SEQ ID NO: 26 and V _L comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises V _H comprising the amino acid sequence of SEQ ID NO: 26 and V _L comprising the amino acid sequence of SEQ ID NO: 27; (5) (i) the Fab comprises V _H comprising the amino acid sequence of SEQ ID NO: 26 and V _L comprising the amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises V _H comprising the amino acid sequence of SEQ ID NO: 28 and V _L comprising the amino acid sequence of SEQ ID NO: 29; (6) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises a _VH comprising an amino acid sequence of SEQ ID NO: 24 and a _VL comprising an amino acid sequence of SEQ ID NO: 25; (7) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 24 and a _VL comprising an amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a _VH comprising an amino acid sequence of SEQ ID NO: 24 and a _VL comprising an amino acid sequence of SEQ ID NO: 25; (8) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 24 and a _VL comprising an amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a VH comprising an amino acid sequence of SEQ ID NO: 26 and _{a VL} comprising an amino acid sequence of SEQ ID NO: or (9) (i) the Fab comprises _{a VH} comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29. 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 _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; (2) (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32; (3) (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 28 and a _VL comprising the amino acid sequence of SEQ ID NO: 29; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34; (4) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises an amino acid sequence of SEQ ID NO: 32; (5) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises an amino acid sequence of SEQ ID NO: 33; (6) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 26 and a _VL comprising an amino acid sequence of SEQ ID NO: 27; and (ii) the scFv comprises an amino acid sequence of SEQ ID NO: 34; (7) (i) the Fab comprises a _VH comprising an amino acid sequence of SEQ ID NO: 24 and a _VL comprising an amino acid sequence of SEQ ID NO: 25 ; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 32; (8) (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 33; or (9) (i) the Fab comprises a _VH comprising the amino acid sequence of SEQ ID NO: 24 and a _VL comprising the amino acid sequence of SEQ ID NO: 25; and (ii) the scFv comprises the amino acid sequence of SEQ ID NO: 34. The expression construct of any one of claims 1 to 42, wherein the construct(s) is a viral vector. The expression construct of claim 43, wherein the viral vector(s) 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. The expression construct of any one of claims 1 to 42, wherein the expression construct comprises a multi-cistronic expression cassette for expressing the caTCR, the CSR and the c-Jun. A multi-cistronic expression construct comprising an expression cassette for expressing: a) a chimeric antibody T cell receptor (TCR) construct (caTCR) comprising: i) an antigen binding module that specifically binds to glypican 3 (GPC3); and ii) a TCR module (TCRM) derived from human γ/δ TCR; b) a chimeric stimulatory receptor (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 derived from the intracellular domain of human CD30; and c) a human c-Jun polypeptide. The expression construct of claim 46, wherein the expression cassette comprises a coding sequence of SEQ ID NO: 1 and: (i) coding sequences of SEQ ID NOs: 28 and 29 and a coding sequence of SEQ ID NO: 33; (ii) coding sequences of SEQ ID NOs: 28 and 29 and a coding sequence of SEQ ID NO: 32; (iii) coding sequences of SEQ ID NOs: 28 and 29 and a coding sequence of SEQ ID NO: 34; (iv) coding sequences of SEQ ID NOs: 26 and 27 and a coding sequence of SEQ ID NO: 32; (v) coding sequences of SEQ ID NOs: 26 and 27 and a coding sequence of SEQ ID NO: 33; (vi) coding sequences of SEQ ID NOs: 26 and 27 and a coding sequence of SEQ ID NO: 34; (vii) coding sequences of SEQ ID NOs: 24 and 25 and a coding sequence of SEQ ID NO: 32; (viii) the coding sequences 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. 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). A performance construct as in any one of claims 46 to 48, wherein the performance cartridge comprises a constitutive or inducible promoter. The expression construct of claim 49, wherein the promoter is the EF-1α promoter. The expression construct of any one of claims 46 to 50, wherein the construct is a viral vector. 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. A recombinant virus comprising the multicistronic expression construct of any one of claims 50 to 52. The recombinant virus of claim 53, wherein the polycistronic expression construct is a lentiviral vector. A method for engineering immune cells, comprising: (a) providing a starting cell population, (b) introducing an expression construct as described in any one of claims 1 to 52 or a recombinant virus as described in claim 53 or 54 into the starting cell population, (c) selecting cells expressing the caTCR, the CSR and the c-Jun as appropriate, and (d) obtaining engineered immune cells from the cells of step (b) or (c). The method of claim 55, wherein the immune cell is a T cell. The method of claim 56, wherein the T cell is selected from the group consisting of: cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells. The method of any one of claims 55 to 57, wherein the starting cell population comprises immune cells. The method of any one of claims 55 to 58, wherein the starting cell population comprises autologous or allogeneic T cells. The method of any one of claims 55 to 57, wherein the starting cell population comprises pluripotent or multipotent cells, and step (d) comprises differentiating the cells of step (b) or (c) into immune cells. A human cell population comprising the expression construct of any one of claims 1 to 52 or the recombinant virus of claim 53 or 54. The human cell population of claim 61, wherein the human cells are immune cells. An immune cell population obtained by the method of any one of claims 55 to 60. The immune cell population of claim 63, wherein the immune cells are human cells. The immune cell of any one of claims 62 to 64, wherein the immune cells are T cells. The immune cell of claim 65, wherein the T cells are CD8 ⁺ T cells. The immune cell of any one of claims 62 to 66, wherein the immune cells express less depletion markers than corresponding cells that do not overexpress c-Jun. 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. 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. A method of killing target cells, comprising contacting the target cells with the pharmaceutical composition of claim 69 under conditions that allow killing of the target cells by immune cells, wherein the target cells express GPC3. The method of claim 70, wherein the immune cells express less depletion markers when in contact with the target cells compared to corresponding immune cells that do not contain the exogenous nucleic acid molecule that causes overexpression of c-Jun. The method of claim 70 or 71, wherein the immune cells are T cells. The method of claim 72, wherein the T cells are CD8 ⁺ T cells. The method of claim 73, wherein the target cells are cancer cells. The method of claim 74, wherein the synergistic stimulatory immune cell signaling module in the CSR is derived from human CD30, and the immune cells express less exhaustion markers than corresponding immune cells engineered to express a CSR whose synergistic stimulatory immune cell signaling module is derived from human CD28. 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. A method for treating a subject in need thereof, comprising administering the pharmaceutical composition of claim 69 to the subject. Use of an expression construct as claimed in any one of claims 1 to 52, a recombinant virus as claimed in claim 53 or 54, or a pharmaceutical composition as claimed in claim 69 for the manufacture of a medicament for treating an individual in need thereof. A 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. The method of claim 77 or the use of claim 78 or 79, wherein the individual suffers from a GPC3-positive disease. The method or use of claim 80, wherein the GPC3-positive disease is cancer. 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. A method for reducing exhaustion of an engineered immune cell, comprising introducing into the engineered immune cell an exogenous nucleic acid molecule that increases the expression of c-Jun in the cell, wherein the engineered immune cell comprises the expression construct of any one of claims 1 to 52.