TW202413646A - Immune cell therapy - Google Patents

Immune cell therapy Download PDF

Info

Publication number
TW202413646A
TW202413646A TW112126959A TW112126959A TW202413646A TW 202413646 A TW202413646 A TW 202413646A TW 112126959 A TW112126959 A TW 112126959A TW 112126959 A TW112126959 A TW 112126959A TW 202413646 A TW202413646 A TW 202413646A
Authority
TW
Taiwan
Prior art keywords
amino acid
seq
acid sequence
gpc3
cells
Prior art date
Application number
TW112126959A
Other languages
Chinese (zh)
Inventor
熊光焰
許少華
泓若 雲
布萊斯 Ｄ 颯特
比讓 Ａ 波爾達吉普爾
瑞秋 Ｃ 琳
舒巴 波特魯裡
肇 平良木
英 王
Original Assignee
美商萊爾免疫藥物股份有限公司
美商優瑞科生物技術公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 美商萊爾免疫藥物股份有限公司, 美商優瑞科生物技術公司 filed Critical 美商萊爾免疫藥物股份有限公司
Publication of TW202413646A publication Critical patent/TW202413646A/en

Links

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

免疫細胞療法Immunocell Therapy

在過去的二十年裏，在免疫學及腫瘤生物學方面的基礎性進展以及大量腫瘤抗原的鑑別促成了基於細胞之免疫療法領域的顯著進展。旨在藉由將自體及離體擴增之T細胞轉移至患者來治療癌症的T細胞療法已引起一些顯著的抗腫瘤反應(Blattman等人, Science. (2004) 305(5681):200-5)。例如，離體擴增的天然存在之腫瘤浸潤淋巴細胞(TIL)的投與調節黑色素瘤患者，包括在涉及肝臟、肺、軟組織及腦之多個部位具有大型侵襲性腫瘤之患者的在50-70%範圍內的客觀反應率(Rosenberg等人, Nat Rev Cancer.(2008) 8(4):299-308; Dudley等人, J Clin Oncol.(2005) 23(10):2346-57)。 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).

TIL療法之廣泛應用的主要限制為難以產生具有抗腫瘤潛力的人類T細胞。作為替代方法，可經由T細胞工程改造將外源性高親和力TCR引入患者之正常自體T細胞中。將此等細胞授受性轉移至淋巴細胞耗乏之患者中已被證明能在諸如黑色素瘤、結腸直腸癌及滑膜肉瘤之癌症中調節腫瘤消退(Kunert等人, Front Immunol. (2013) 4:363; Robbins等人, Clin Cancer Res. (2015) 21(5):1019-27)。經TCR工程改造之T細胞療法的優勢之一在於其可靶向潛在胞內腫瘤特異性蛋白質之整個陣列，該等蛋白質經由MHC呈遞處理及遞送至細胞表面；此等抗原甚至可在較低密度下被抗原特異性細胞毒性T細胞識別到(Kunert，見上文)。 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).

已作出若干嘗試來工程改造具有抗體特異性與T細胞受體效應功能的TCR分子。在此等方法中之一些中，TCR (例如αβ TCR或γδ TCR)之可變域及恆定域經針對腫瘤抗原之抗體的可變域及恆定域置換，從而產生嵌合抗體TCR，稱作「abTCR」或「caTCR」。參見例如WO 2017/070608及WO 2018/200582，其揭示內容以全文引用之方式併入本文中。在該等方法中之一者中，嵌合刺激受體(CSR)與caTCR組合使用以增強經工程改造之T細胞的腫瘤殺傷功效。如同嵌合抗原受體(CAR)，CSR具有結合目標配位體(例如腫瘤抗原)之胞外域以及胞內協同刺激域，但不同於CAR，CSR不具有胞內初級免疫細胞信號傳導域(其通常為CD3 ζ鏈的胞內域)或功能性初級免疫細胞信號傳導域。CSR及caTCR可結合同一目標/抗原之不同抗原決定基且協同作用以增強經工程改造之T細胞的細胞毒性。參見例如WO 2018/200583，其揭示內容以全文引用之方式併入本文中。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.

T細胞療法面臨之一個挑戰為由於一種稱為T細胞耗竭之現象，T細胞在活體內缺乏持久性。參見例如Fraietta等人, Nat Med. (2018) 24(5):563-71; Long等人, Nat Med. (2015) 21(6):581-90; 及Eyquem等人, Nature(2017) 543(7643):113-7。T細胞耗竭之特徵為代謝功能顯著變化、轉錄程式化、效應功能喪失(例如細胞介素分泌及細胞毒性降低)、多種表面抑制受體表現及細胞凋亡。T細胞耗竭歸因於恆定的抗原暴露，導致持續的TCR信號傳導，或經由T細胞上之經工程改造之抗原受體的強直性抗原獨立信號傳導(參見例如Long，同上)。在例如患有癌症或慢性感染之患者中及在T細胞療法中，已尋求預防或逆轉T細胞耗竭作為增強T細胞有效性之手段。參見例如WO 2019/118902，其揭示內容以全文引用的方式併入本文中。 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.

磷脂醯肌醇蛋白聚糖3 (GPC3，亦稱為SGB、DGSX、MXR7、SDYS、SGBS、OCI-5、SGBS1、GTR2-2)，係一種在多種癌症類型中過度表現的細胞表面蛋白，該等癌症包括多種實體腫瘤，諸如肝細胞癌(HCC)、黑色素瘤(Nakatsura T等人, Clin Cancer Res .(2004) 10(19):6612-21)、肺部鱗狀細胞癌(Yu X等人, Genet Mol Res .(2015) 14(3):10185-92)、卵巢癌(Stadlmann S等人, Int J Gynecol Pathol .(2007) 26(3):341-4)、卵黃囊瘤、絨膜癌(Zynger DL等人, Am J Surg Pathol .(2006) 30(12):1570-5)、威爾姆斯氏瘤(Wilms' tumor)及脂肪肉瘤(Baumhoer D等人, Am J Clin Pathol .(2008) 129(6):899-906)。醫學上仍需要開發針對各種癌症(包括過度表現GPC3之癌症)的療法。 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.

因此，仍需要其中經工程改造之T細胞具有較高且持續的腫瘤殺傷效能的改良之T細胞療法，且尤其是其中T細胞特異性靶向過度表現GPC3之癌症的改良之T細胞療法。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.

本發明提供用於改良免疫細胞療法之組合物及方法。在一些態樣中，本文提供一或多種表現構築體，其包含一或多個用於表現以下之表現卡匣：a)嵌合抗體T細胞受體(TCR)構築體(caTCR)，其包含：i)特異性結合磷脂醯肌醇蛋白聚糖3 (GPC3)的抗原結合模組；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)嵌合刺激受體(CSR)，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。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.

在一些實施例中，c-Jun為野生型人類c-Jun。在一些實施例中，野生型人類c-Jun與SEQ ID NO: 1之胺基酸序列具有至少約90%一致性。在一些實施例中，c-Jun為突變型人類c-Jun。在一些實施例中，突變型人類c-Jun在其反式活化域或δ域中包含不活化突變。在一些實施例中，c-Jun包含：(i)相比於SEQ ID NO: 1之S63A及S73A取代；或(ii)相比於SEQ ID NO: 1之胺基酸殘基2與102之間或胺基酸殘基30與50之間的缺失。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.

在一些實施例中，GPC3為細胞表面所結合之GPC3。In some embodiments, GPC3 is cell surface-bound GPC3.

在一些實施例中，TCRM源於人類γ/δ TCR。在一些實施例中，caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一及第二穩定域彼此具有使該caTCR穩定的結合親和力。在一些實施例中，穩定模組係選自由以下組成之群：TCR恆定域、C H1-C L模組、C H2-C H2模組、C H3-C H3模組及C H4-C H4模組。在一些實施例中，穩定模組係衍生自人類蛋白質。在一些實施例中，該C H1-C L模組中所含之C L係衍生自κ抗體輕鏈或λ抗體輕鏈。在一些實施例中，caTCR之抗原結合模組為Fab、Fab'、(Fab') 2、Fv或單鏈Fv (scFv)。 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).

在一些實施例中，caTCR之抗原結合模組包含：(i)包含有包含SEQ ID NO: 6之胺基酸序列的重鏈互補決定區(HC-CDR) 1 (HC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的重鏈可變域(V H)；及包含有包含SEQ ID NO: 9之胺基酸序列的輕鏈互補決定區(LC-CDR) 1 (LC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含GDN之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的輕鏈可變域(V L)；(ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含YDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L；或(iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含DDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L。在一些實施例中，caTCR之抗原結合模組包含：(i)包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L；(ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；或(iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。 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.

在一些實施例中，caTCR之抗原結合模組包含：(i)包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25具有至少約90%序列一致性之胺基酸序列的V L；(ii)包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27具有至少約90%序列一致性之胺基酸序列的V L；或(iii)包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29具有至少約90%序列一致性之胺基酸序列的V L。在一些實施例中，caTCR之抗原結合模組包含：(i)包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；(ii)包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；或(iii)包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 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.

在一些實施例中，caTCR為雜二聚體，其包含有包含第一TCRD之第一多肽鏈及包含第二TCRD之第二多肽鏈。在一些實施例中，caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V L。在一些實施例中，caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V L。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 30之胺基酸序列或與SEQ ID NO: 30具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 31之胺基酸序列或與SEQ ID NO: 31具有至少約90%序列一致性之胺基酸序列。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 10之胺基酸序列或與SEQ ID NO: 10具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 5之胺基酸序列或與SEQ ID NO: 5具有至少約90%序列一致性之胺基酸序列。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 63之胺基酸序列或與SEQ ID NO: 63具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 64之胺基酸序列或與SEQ ID NO: 64具有至少約90%序列一致性之胺基酸序列。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 65之胺基酸序列或與SEQ ID NO: 65具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 66之胺基酸序列或與SEQ ID NO: 66具有至少約90%序列一致性之胺基酸序列。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 67之胺基酸序列或與SEQ ID NO: 67具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 68之胺基酸序列或與SEQ ID NO: 68具有至少約90%序列一致性之胺基酸序列。在一些實施例中，caTCR為雜二聚體，其包含：第一多肽鏈，其包含SEQ ID NO: 69之胺基酸序列或與SEQ ID NO: 69具有至少約90%序列一致性之胺基酸序列；及第二多肽鏈，其包含SEQ ID NO: 70之胺基酸序列或與SEQ ID NO: 70具有至少約90%序列一致性之胺基酸序列。 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.

在一些實施例中，CSR之跨膜模組包含衍生自CD30、CD28、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137或CD154之跨膜域的跨膜域。在一些實施例中，CSR之跨膜模組包含衍生自CD30之跨膜域的跨膜域。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.

在一些實施例中，協同刺激免疫細胞信號傳導模組衍生自TCR之協同刺激受體的胞內域。在一些實施例中，協同刺激受體係選自由CD30、CD28、4-IBB、OX40、ICOS、CD27及CD40組成之群。在一些實施例中，CSR之協同刺激免疫細胞信號傳導模組衍生自人類CD30。在一些實施例中，人類CD30之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 43之胺基酸序列。在一些實施例中，人類CD30之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 44之胺基酸序列。在一些實施例中，CSR之協同刺激免疫細胞信號傳導模組及跨膜域均源於CD30 (例如人類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).

在一些實施例中，CSR之配位體結合模組包含：(i)包含有包含SEQ ID NO: 6之胺基酸序列的重鏈互補決定區(HC-CDR) 1 (HC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的重鏈可變域(V H)；及包含有包含SEQ ID NO: 9之胺基酸序列的輕鏈互補決定區(LC-CDR) 1 (LC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含GDN之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的輕鏈可變域(V L)；(ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含YDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L；或(iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含DDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L。在一些實施例中，CSR之配位體結合模組包含：(i)包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L；(ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；或(iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。 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.

在一些實施例中，CSR之配位體結合模組包含：(i)包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25具有至少約90%序列一致性之胺基酸序列的V L；(ii)包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27具有至少約90%序列一致性之胺基酸序列的V L；或(iii)包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29具有至少約90%序列一致性之胺基酸序列的V L。在一些實施例中，CSR之配位體結合模組包含：(i)包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；(ii)包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；或(iii)包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 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.

在一些實施例中，配位體結合模組CSR包含：(i) SEQ ID NO: 32或與SEQ ID NO: 32具有至少約90%序列一致性之胺基酸序列；(ii) SEQ ID NO: 33或與SEQ ID NO: 33具有至少約90%序列一致性之胺基酸序列；或(iii) SEQ ID NO: 34或與SEQ ID NO: 34具有至少約90%序列一致性之胺基酸序列。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.

在一些實施例中，(i) caTCR之抗原結合模組包含：(a)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及(b)包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L；且(ii) CSR之配位體結合模組包含：(a)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及(b)包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L。 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.

在一些實施例中，caTCR之抗原結合模組包含結合GPC3之Fab，其中該CSR之配位體結合模組包含結合GPC3之scFv，且其中該CSR之跨膜模組及該協同刺激免疫細胞信號傳導模組均衍生自CD30 (例如人類CD30)。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).

在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 In some embodiments, the CSR comprises a fragment of CD30 comprising the amino acid sequence of SEQ ID NO: 43, and (i) the Fab comprises a 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.

在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 32之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 34之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 32之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 34之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 32之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，CSR包含有包含SEQ ID NO: 43之胺基酸序列的CD30之片段，且(i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及(ii)該scFv包含SEQ ID NO: 34之胺基酸序列。 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.

在一些實施例中，構築體為病毒載體。在一些實施例中，病毒載體係選自由以下組成之群：慢病毒載體、腺病毒載體、腺相關病毒載體、牛痘載體、單純疱疹病毒載體及EB病毒載體(Epstein-Barr viral vector)。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.

在一些實施例中，表現構築體包含用於表現caTCR、CSR及c-Jun之多順反子表現卡匣。In some embodiments, the expression construct comprises a polycistronic expression cassette for expressing caTCR, CSR, and c-Jun.

在一些態樣中，本文提供一種多順反子表現構築體，其包含用於表現以下之表現卡匣：a)嵌合抗體T細胞受體(TCR)構築體(caTCR)，其包含：i)特異性結合磷脂醯肌醇蛋白聚糖3 (GPC3)的抗原結合模組；及ii)衍生自人類γ/δ TCR之TCR模組(TCRM)；b)嵌合刺激受體(CSR)，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)衍生自人類CD30之胞內域的協同刺激免疫細胞信號傳導模組；及c)人類c-Jun多肽。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 32之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 34之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 32之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 34之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 32之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 34之編碼序列。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.

在一些實施例中，編碼序列在框內藉由2A編碼序列或藉由內部核糖體進入位點(IRES)分開。在一些實施例中，表現卡匣包含組成性或可誘導型啟動子。在一些實施例中，啟動子為EF-1α啟動子。在一些實施例中，構築體為病毒載體。在一些實施例中，病毒載體係選自由以下組成之群：慢病毒載體、腺病毒載體、腺相關病毒載體、牛痘載體、單純疱疹病毒載體及EB病毒載體。在一些實施例中，本文提供一種重組病毒，其包含如前述實施例中任一者之多順反子表現構築體。在一些實施例中，多順反子表現構築體為慢病毒載體。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.

在一些態樣中，本文提供一種工程改造免疫細胞之方法，其包含：(a)提供起始細胞群；(b)將如前述技術方案中任一項之表現構築體或如前述技術方案中任一項之重組病毒引入起始細胞群中；(c)視情況選擇表現caTCR、CSR及c-Jun之細胞；及(d)自步驟(b)或(c)之細胞獲得經工程改造之免疫細胞。在一些實施例中，免疫細胞為T細胞。在一些實施例中，T細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，起始細胞群包含免疫細胞。在一些實施例中，起始細胞群包含自體或同種異體T細胞。在一些實施例中，起始細胞群包含多能或多潛能細胞，且步驟(d)包含使步驟(b)或(c)之細胞分化成免疫細胞。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.

在一些態樣中，本文提供一種人類細胞群，其包含前述實施例中任一者之表現構築體或前述實施例中任一者之重組病毒。在一些實施例中，人類細胞為免疫細胞。在一些實施例中，免疫細胞群係藉由如前述實施例中任一者之方法獲得。在一些實施例中，免疫細胞為人類細胞。在一些實施例中，免疫細胞為T細胞。在一些實施例中，T細胞為CD8 +T細胞。在一些實施例中，免疫細胞相比於不過度表現c-Jun之對應細胞，表現較少量之耗竭標記物。在一些實施例中，耗竭標記物係選自由CD39、PD-1、TIM-3及LAG-3組成之群。 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.

在一些態樣中，本文提供一種醫藥組合物，其包含如前述實施例中任一者之表現構築體、如前述實施例中任一者之重組病毒或如前述實施例中任一者之細胞及醫藥學上可接受之載劑。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.

在一些態樣中，本文提供一種殺傷目標細胞之方法，其包含使目標細胞與如前述實施例中任一者之醫藥組合物在允許由免疫細胞殺傷目標細胞之條件下接觸，其中目標細胞表現GPC3。在一些實施例中，免疫細胞相比於不包含引起c-Jun過度表現之外源性核酸分子的對應免疫細胞，在與目標細胞接觸時表現較少量之耗竭標記物。在一些實施例中，免疫細胞為T細胞。在一些實施例中，T細胞為CD8 +T細胞。在一些實施例中，目標細胞為癌細胞。在一些實施例中，CSR中之協同刺激免疫細胞信號傳導模組衍生自人類CD30，且該等免疫細胞相比於經工程改造以表現其中之協同刺激免疫細胞信號傳導模組源於人類CD28的CSR的對應免疫細胞，表現較少量之耗竭標記物。在一些實施例中，耗竭標記物係選自由CD39、PD-1、TIM-3及LAG-3組成之群。 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.

在一些態樣中，本文提供一種治療有需要之個體的方法，其包含向該個體投與如前述實施例中任一者之醫藥組合物。在一些態樣中，本文提供前述實施例中之任一者之表現構築體、前述實施例中之任一者之重組病毒或前述實施例中之任一者之醫藥組合物的用途，其用於製造供治療有需要之個體用之藥劑。在一些態樣中，本文提供前述實施例中之任一者之表現構築體、前述實施例中之任一者之重組病毒或前述實施例中之任一者之醫藥組合物的用途，其用於治療有需要之個體。在一些實施例中，個體患有GPC3陽性疾病。在一些實施例中，GPC3陽性疾病為癌症。在一些實施例中，癌症係選自由以下組成之群：肝細胞癌(HCC)、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。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.

在一些態樣中，本文提供一種減少經工程改造之免疫細胞耗竭的方法，其包含向該經工程改造之免疫細胞中引入增加該細胞中c-Jun之表現的外源性核酸分子，其中該經工程改造之免疫細胞包含如前述實施例中任一者之表現構築體。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.

相關申請案之交叉引用Cross-references to related applications

本申請案主張2022年7月22日申請之美國臨時申請案63/369,171之優先權。前述臨時申請案之揭示內容以全文引用之方式併入本文中。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.

本申請案提供包含用於表現嵌合抗體T細胞受體(caTCR)、嵌合刺激受體(CSR；在本文中亦被稱為「嵌合信號傳導受體」)及c-Jun多肽(例如人類c-Jun多肽)之構築體的經工程改造之人類細胞(例如免疫細胞，諸如T細胞)。caTCR與CSR均特異性結合GPC3或與其相互作用(本文中亦分別稱為「抗GPC3 caTCR」及「抗GPC3 CSR」)。抗GPC3 caTCR包含特異性結合GPC3的抗原結合模組，及能夠募集至少一種TCR相關信號傳導分子的T細胞受體模組(TCRM)。抗GPC3 CSR包含特異性結合GPC3之配位體結合域，及能夠提供刺激信號至免疫細胞且不包含功能性初級T細胞信號傳導序列的協同刺激免疫細胞信號傳導域。本文所提供之抗GPC3 caTCR及抗GPC3 CSR所靶向的GPC3可表現於目標細胞(例如病變細胞)之細胞表面上。在一些實施例中，抗GPC3 caTCR及抗GPC3 CSR結合同一GPC3蛋白質上的不同區域。該疾病可為癌症，諸如胃腸道系統癌症，例如胃癌或肝癌。抗GPC3 caTCR係藉由控制TCR活化的天然存在之機制來調節，而抗GPC3 CSR增強由抗GPC3 caTCR介導的免疫反應。c-Jun表現有助於藉由例如緩解或阻止T細胞耗竭來維持T細胞之活性狀態。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.

諸如T細胞之本發明之經工程改造之免疫細胞針對攜帶目標之腫瘤細胞展現出持續有效的細胞毒性。相比於不過度表現c-Jun (例如經由編碼c-Jun之外源性引入之核酸序列)之T細胞，本發明之經工程改造之T細胞顯示較少T細胞耗竭跡象。經工程改造之細胞可具有以下特徵中之一或多者：(i)其不會隨時間推移而具有增加的耗竭標記物PD-1、TIM-3及/或LAG-3表現量；(ii)具有降低的細胞凋亡率；(iii)其增加形成記憶細胞及/或維持記憶標記物(例如CCR7及CD45RA)；(iv)其具有增強的細胞毒性；(v)其顯示提高的對具有低表面抗原之腫瘤目標的識別；(vi)其具有增強的回應於抗原之增殖；(vii)在重複抗原刺激之後維持存活及功能；及(viii)其顯示增加的腫瘤浸潤能力。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.

在一些實施例中，抗GPC3 CSR包含源於CD30 (例如人類CD30)之胞內域的協同刺激免疫細胞信號傳導域。本發明人意外地發現，c-Jun過度表現顯著減少經工程改造以表現抗GPC3 caTCR及基於CD30之抗GPC3 CSR之T細胞的耗竭。 I. 定義 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

術語「抗體」或「抗體部分」包括全長抗體及其抗原結合片段。全長抗體包含兩條重鏈及兩條輕鏈。輕鏈及重鏈之可變區(亦即可變域)負責抗原結合。兩種鏈中之可變區一般均含有三個稱作互補決定區(CDR)之高度可變迴路(輕鏈(LC)CDR包括LC-CDR1、LC-CDR2及LC-CDR3，重鏈(HC)CDR包括HC-CDR1、HC-CDR2及HC-CDR3)。本文所揭示之抗體及抗原結合片段的CDR邊界可根據Kabat、Chothia或Al-Lazikani公約(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)界定或鑑別。重鏈或輕鏈之三個CDR***稱為構架區(FR)之側接片段之間，該等側接片段的保守性比CDR更高且形成支撐高變環之支架。重鏈及輕鏈之恆定區不涉及抗原結合，但展現多種效應功能。抗體基於其重鏈恆定區之胺基酸序列分類。抗體之五種主要類別或同型為IgA、IgD、IgE、IgG及IgM，其特徵為分別存在α、δ、ε、γ及μ重鏈。若干主要抗體類別劃分成如下子類，諸如lgG1 (γ1重鏈)、lgG2 (γ2重鏈)、lgG3 (γ3重鏈)、lgG4 (γ4重鏈)、lgA1 (α1重鏈)或lgA2 (α2重鏈)。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).

如本文所用之術語「抗原結合片段」係指抗體片段，包括例如雙功能抗體、Fab、Fab'、F(ab')2、Fv片段、二硫鍵穩定之Fv片段(dsFv)、(dsFv)2、雙特異性dsFv (dsFv-dsFv')、二硫鍵穩定之雙功能抗體(ds雙功能抗體)、單鏈抗體分子(scFv)、scFv二聚體(二價雙功能抗體)、由包含一或多個CDR之抗體的一部分形成之多特異性抗體、駱駝的單域抗體、奈米抗體、域抗體、二價域抗體、或結合抗原但不包含完整抗體結構之任何其他抗體片段。抗原結合片段能夠結合與親本抗體或親本抗體片段(例如親本scFv)所結合相同之抗原。在一些實施例中，抗原結合片段可包含移植至來自一或多種不同人類抗體之構架區的來自特定人類抗體之一或多種CDR。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.

如本文所用，術語「特異性結合」或「對……具有特異性」係指可量測及可再現相互作用，如目標與抗體或抗體部分之間的結合，其在非均質分子(包括生物分子)群體存在下決定目標之存在。舉例而言，特異性結合目標(其可為抗原決定基)之抗體部分係與目標結合之親和力、親合力、容易性及/或持續時間大於其結合其他目標之抗體部分。在一些實施例中，特異性結合抗原之抗體部分與抗原(例如細胞表面抗原或肽/MHC蛋白質複合體)之一或多個抗原決定子之反應的結合親和力係其針對其他目標之結合親和力的至少約10倍。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.

術語「T細胞受體」或「TCR」係指由T細胞表面上配對的αβ或γδ鏈構成之雜二聚體受體。各α、β、γ及δ鏈由兩個Ig樣域構成：經由互補決定區(CDR)賦予抗原識別之可變域(V)，隨後為藉由連接肽及跨膜(TM)區錨定至細胞膜的恆定域(C)。TM區與CD3信號傳導設備之恆定次單位結合。V域中之各者具有三個CDR。此等CDR與結合由主要組織相容性複合物(pMHC)編碼之蛋白質的抗原肽之間的複合物相互作用(Davis及Bjorkman (1988) Nature, 334, 395-402；Davis等人 (1998) Annu Rev Immunol, 16, 523-544；Murphy (2012), xix, 第868頁)。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).

術語「TCR相關信號傳導分子」係指具有基於細胞質免疫受體酪胺酸之活化模體(ITAM)的分子，其為TCR-CD3複合體之一部分。TCR相關信號傳導分子包括CD3γε、CD3δε及ζζ (亦稱為CD3ζ或CD3ζζ)。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).

如本文關於表現CD3之細胞所用的「活化」係指已經足夠刺激以誘發CD3信號傳導路徑之下游效應功能(包括但不限於細胞增殖及細胞介素產生)的可偵測提高的細胞之狀態。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.

術語「模組」在涉及蛋白質之一部分時意謂包括構成蛋白質之一或多種多肽之結構上及/或功能上相關之部分。舉例而言，二聚體受體之跨膜模組可指受體之每一多肽鏈之跨膜部分。模組亦可指單一多肽鏈之相關部分。舉例而言，單體受體之跨膜模組可指受體之單一多肽鏈之跨膜部分。模組亦可僅包括多肽之單一部分。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.

如本文所用，術語「分離之核酸」意指基因體、cDNA或合成來源或其某一組合之核酸，藉助於其來源，「分離之核酸」(1)不與自然界中該「分離之核酸」所存在之聚核苷酸之全部或一部分結合，(2)可操作地連接於自然界中不與其連接之聚核苷酸，或(3)在自然界中不作為較大序列之一部分存在。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.

如本文所用，術語「CDR」或「互補決定區」意指重鏈與輕鏈多肽之可變區內發現的非鄰接抗原組合位點。此等特定區已由Kabat等人, J. Biol. Chem.252:6609-6616 (1977)；Kabat等人, U.S. Dept. of Health and Human Services, 「Sequences of proteins of immunological interest」 (1991)；Chothia等人, J. Mol. Biol.196:901-917 (1987)；Al-Lazikani B.等人, J. Mol. Biol., 273: 927-948 (1997)；MacCallum等人, J. Mol. Biol.262:732-745 (1996)；Abhinandan及Martin, Mol. Immunol., 45: 3832-3839 (2008)；Lefranc M.P.等人, Dev. Comp. Immunol., 27: 55-77 (2003)；及Honegger及Plückthun, J. Mol. Biol., 309:657-670 (2001)描述，其中定義包括當彼此比較時胺基酸殘基之重疊或子集。然而，應用任一定義來提及抗體或所移植抗體之CDR或其變異體均意欲屬於如本文所定義及使用之術語的範疇內。涵蓋以上所引用參考文獻中之各者所定義的CDR的胺基酸殘基如下闡述於 表 1中作為對比物。CDR預測演算法及界面為此項技術中已知的，包括例如Abhinandan及Martin, Mol. Immunol., 45: 3832-3839 (2008)；Ehrenmann F.等人, Nucleic Acids Res., 38: D301-D307 (2010)；及Adolf-Bryfogle J.等人, Nucleic Acids Res., 43: D432-D438 (2015)。此段落中所引用之參考文獻的內容以全文引用的方式併入本文中以用於本發明中且可能包括於本文的一或多個技術方案中。本發明之特定CDR序列由IMGT編號系統定義。 表 1. CDR 定義 .    Kabat 1 Chothia 2 MacCallum 3 IMGT 4 AHo 5 V HCDR1 31-35 26-32 30-35 27-38 25-40 V HCDR2 50-65 53-55 47-58 56-65 58-77 V HCDR3 95-102 96-101 93-101 105-117 109-137 V LCDR1 24-34 26-32 30-36 27-38 25-40 V LCDR2 50-56 50-52 46-55 56-65 58-77 V LCDR3 89-97 91-96 89-96 105-117 109-137 1殘基編號遵循上述Kabat等人之命名法 2殘基編號遵循上述Chothia等人之命名法 3殘基編號遵循上述MacCallum等人之命名法 4殘基編號遵循上述Lefranc等人之命名法 5殘基編號遵循上述Honegger及Plückthun之命名法 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

術語「嵌合抗體」係指其中重鏈及/或輕鏈的一部分與源於特定物種或屬於特定抗體類別或子類之抗體中的對應序列一致或同源，而該鏈中的其餘部分與源於另一物種或屬於另一抗體類別或子類之抗體中的對應序列一致或同源的抗體，以及此類抗體之片段，只要其展現本發明之生物活性(參見美國專利第4,816,567號；及Morrison等人, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984))。 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)).

關於抗體或抗體部分之術語「半合成」意指抗體或抗體部分具有一或多個天然存在之序列及一或多個非天然存在之(亦即合成)序列。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.

關於抗體或抗體部分之術語「全合成」意謂抗體或抗體部分具有固定的天然存在之V H/V L構架配對，而非重鏈及輕鏈兩者之所有6種CDR之非天然存在之(亦即合成)序列。 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」為含有完整抗原識別及結合位點之最小抗體片段。此片段由緊密、非共價締合之一個重鏈可變區域與一個輕鏈可變區域之二聚體組成。此兩個域摺疊得到六個高變環(重鏈及輕鏈各有3個環)，其貢獻了胺基酸殘基用於抗原結合且向抗體賦予抗原結合特異性。然而，即使單一可變域(或僅包含三個對抗原具有特異性之CDR之Fv的一半)能夠辨識且結合抗原，但親和力比整個結合位點低。"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.

「單鏈Fv」(亦縮寫為「sFv」或「scFv」)為包含連接於單一多肽鏈中之V H及V L抗體域的抗體片段。在一些實施例中，scFv多肽進一步包含介於V H與V L域之間的多肽連接子，其使得scFv能夠形成用於抗原結合的所需結構。關於scFv之評述，參見Plückthun, 於 The Pharmacology of Monoclonal Antibodies, 第113卷, Rosenburg及Moore編, Springer-Verlag, New York, 第269-315頁(1994)中。 "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).

非人類(例如嚙齒動物)抗體之「人源化」形式為含有源於非人類抗體之最小序列的嵌合抗體。在很大程度上，人源化抗體為人類免疫球蛋白(接受者抗體)，其中來自接受者的高變區(HVR)殘基經來自非人類物種(供體抗體) (諸如具有所期望抗體特異性、親和力及能力之小鼠、大鼠、兔或非人類靈長類動物)的高變區之殘基置換。在一些情況下，人類免疫球蛋白之構架區(FR)殘基經對應非人類殘基置換。此外，人源化抗體可包含在接受者抗體或供體抗體中未發現之殘基。進行此等修飾以進一步改進抗體效能。一般而言，人源化抗體將包含至少一個且通常兩個可變域中的實質上所有可變域，其中所有或實質上所有高變環對應於非人類免疫球蛋白之高變環且所有或實質上所有FR為人類免疫球蛋白序列之FR。人源化抗體視情況亦將包含免疫球蛋白恆定區(Fc)之至少一部分，通常，人類免疫球蛋白之恆定區的至少一部分。 關於其他細節，參見 Jones 等人 , Nature 321:522-525 (1986); Riechmann 等人 , Nature 332:323-329 (1988); 及 Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) 。 "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) .

「同源性」係指兩個多肽之間或兩個核酸分子之間的序列相似性或序列一致性。當所比較的兩個序列中之一個位置由相同鹼基或胺基酸單體次單元佔據時，例如若兩個DNA分子中之各者中的一個位置由腺嘌呤佔據，則該等分子在該位置處「同源」。兩個序列之間的「同源性百分比」或「序列一致性百分比」為兩個序列共用之匹配或同源位置之數目除以所比較位置之數目乘以100之函數，任何保守取代視為序列一致性之一部分。舉例而言，若兩個序列中之10個位置中有6個匹配或同源，則兩個序列為60%同源。舉例而言，DNA序列ATTGCC與TATGGC共用50%同源性。一般而言，在比對兩個序列以得到最大同源性時進行了比較。用於測定胺基酸序列一致性百分比之目的之比對可以此項技術中之技能範圍內的各種方式實現，例如使用公開可獲得的電腦軟體，如BLAST、BLAST-2、ALIGN、Megalign (DNASTAR)或MUSCLE軟體。熟習此項技術者可確定用於量測比對之適當參數，包括用於實現所比較序列之全長內之最大比對所需的任何演算法。然而，出於本文之目的，使用序列比較電腦程式MUSCLE產生胺基酸序列一致性%值(Edgar, R.C., Nucleic Acids Research32(5):1792-1797, 2004；Edgar, R.C., BMC Bioinformatics5(1):113, 2004)。 "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).

除非另外指定，否則「編碼胺基酸序列之核苷酸序列」包括為彼此之簡併型式且編碼相同胺基酸序列之所有核苷酸序列。就編碼蛋白質之核苷酸序列可以在一些形式中含有內含子而言，片語編碼該蛋白質或RNA之核苷酸序列亦可包括內含子。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.

術語「可操作地連接」係指調節序列與異源核酸序列之間的功能連接，從而引起後者表現。舉例而言，當第一核酸序列與第二核酸序列處於官能性關係時，第一核酸序列可操作地連接第二核酸序列。舉例而言，若啟動子影響編碼序列之轉錄或表現，則啟動子可操作地連接於編碼序一般而言，可操作地連接之DNA序列為鄰接的，且必要時使兩個蛋白質編碼區在同一個讀框中連接。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.

術語「誘導型啟動子」係指可藉由添加或移除一或多個特定信號來調節活性的啟動子。舉例而言，誘導型啟動子可在一組特定條件下活化可操作地連接之核酸的轉錄，例如在活化啟動子及/或緩解啟動子之壓製的誘導劑或條件存在下。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.

如本文所用，術語「細胞工程改造」或「細胞修飾」(包括其衍生物)係指細胞，例如本文揭示之免疫細胞之靶向修飾。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.

如本文所用，「治療(treatment)」或「治療(treating)」為用於獲得有益或期望結果(包括臨床結果)之方法。出於本發明之目的，有益或所期望臨床結果包括但不限於以下中之一或多者：緩解疾病所引起之一或多種症狀、縮小疾病範圍、穩定疾病(例如預防或延緩疾病惡化)、預防或延緩疾病擴散(例如轉移)、預防或延緩疾病復發、延緩或減緩疾病惡化、改善疾病狀態、使得疾病緩解(部分或完全)、減少治療疾病所需的一或多種其他藥劑之劑量、延緩疾病惡化、提高或改善生活品質、增加體重增加量及/或延長存活期。「治療(treatment)」亦涵蓋減少疾病之病理結果(諸如癌症中之腫瘤體積)。本發明之方法涵蓋該等治療態樣中之任一者或多者。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.

術語「復發(recurrence)」、「復發(relapse)」或「復發(relapsed)」係指癌症或疾病在疾病消失之臨床評估之後的恢復。遠端癌轉移或局部復發之診斷可視為復發。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.

術語「難治性」或「耐受性」係指不回應於治療之癌症或疾病。The term "refractory" or "resistant" refers to a cancer or disease that does not respond to treatment.

如本文所揭示之caTCR或包含caTCR之組合物的「有效量」係足以進行特定陳述之目的之量。「有效量」可憑經驗及藉由與所述目的相關之已知方法確定。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.

術語「治療有效量」係指有效地「治療」個體之疾病或病症的如本文所揭示之caTCR或包含caTCR之組合物的量。在癌症之情況下，治療有效量之如本文所揭示之caTCR或包含caTCR之組合物可減少癌細胞數目；減小腫瘤尺寸或重量；抑制(亦即在一定程度上減緩且較佳阻止)癌細胞浸潤至周邊器官中；抑制(亦即在一定程度上減緩且較佳阻止)腫瘤轉移；在一定程度上抑制腫瘤生長；及/或在一定程度上緩解一或多種與癌症相關之症狀。就如本文所揭示之caTCR或包含caTCR之組合物可阻止生長及/或殺傷現有癌細胞而言，其可為細胞生長抑制及/或細胞毒性的。在一些實施例中，治療有效量為生長抑制量。在一些實施例中，治療有效量為提高患者之無進展存活期的量。在諸如病毒感染之感染性疾病之情況下，治療有效量之如本文所揭示之caTCR或包含caTCR之組合物可減少感染病原體之細胞數目；減少源自病原體之抗原之產生或釋放；抑制(亦即在一定程度上減緩且較佳阻止)病原體擴散至未感染細胞；及/或在一定程度上緩解一或多種與感染相關之症狀。在一些實施例中，治療有效量為延長患者存活期之量。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.

如本文所用，「醫藥學上可接受」或「藥理學上相容」意謂在生物學上或在其他方面無不良影響之材料，亦即，該材料可併入投與患者之醫藥組合物中而不會引起任何顯著不良生物學效應或以有害方式與含有其之組合物中的任何其他組分相互作用。醫藥學上可接受之載劑或賦形劑較佳滿足毒理學及製造測試之必需標準及/或包括於美國食品藥物管理局(U.S. Food and Drug administration)制定之非活性成分指南(Inactive Ingredient Guide)中。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.

應瞭解，本文所述之本發明實施例包括「由實施例組成」及/或「基本上由實施例組成」。It should be understood that the embodiments of the present invention described herein include "consisting of" and/or "consisting essentially of" the embodiments.

本文中對「約」值或參數之提及包括(及描述)針對該值或參數本身之變化形式。舉例而言，提及「約X」之描述包括對「X」之描述。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."

如本文所用，提及「不為一值或參數」一般意謂及描述「除一值或參數外」。舉例而言，方法不用於治療X型癌症意謂該方法用於治療除X型外的類型之癌症。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.

除非上下文另外明確指示，否則如本文中及所附申請專利範圍中所使用，單數形式「一」、「或」及「該」包括複數個指示物。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.

除非本文另外定義，否則結合本發明使用之科學與技術術語將具有一般熟習技術者通常理解之含義。儘管可使用類似於或等效於本文中所描述之彼等的方法及材料來實踐或測試本發明，但下文描述例示性方法及材料。在有矛盾的情況下，將以本發明(包括定義)為準。一般而言，與本文所述之免疫學、醫學、藥物及醫藥化學以及細胞生物學結合使用之命名法及其技術為此項技術中熟知且常用者。此外，除非上下文另外需要，否則單數術語應包括複數術語且複數術語應包括單數術語。在本說明書及實施例通篇中，詞語「具有(have)」及「包含(comprise)」或諸如「具有(has)」、「具有(having)」、「包含(comprises)」或「包含(comprising)」之變化形式應理解為暗示包括陳述的整數或整數群，但不排除任何其他整數或整數群。本文所提及之所有公開案及其他參考文獻均以全文引用之方式併入本文中。儘管本文中引用多個文件，但此引用不構成此等文件中之任一者形成此項技術中公共常識之部分的許可。如本文所用，術語「大致」或「約」當應用於所關注之一或多個值時係指類似於所陳述參考值的值。在某些實施例中，除非另外說明或另外自上下文顯而易見，否則該術語係指在陳述參考值之任一方向(大於或小於)上在10%、9%、8%、7%、6%、5%、4%、3%、2%、1%或小於1%內的值範圍。 II. 表現構築體 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

本發明提供一或多種表現構築體，其包含一或多個表現卡匣，該等表現卡匣用於根據本文中所描述之caTCR (例如caTCR，其包含特異性結合GPC3之抗原結合模組)、CSR (例如包含能夠與GPC3結合或相互作用之配位體結合模組的CSR)及c-Jun多肽(例如人類c-Jun多肽)，表現caTCR、CSR及c-Jun多肽(該(一或多種)表現構築體在本文中亦被稱為「(一或多種) caTCR+CSR+c-Jun表現構築體」)。本發明之caTCR及CSR特異性靶向GPC3 (此類caTCR及CSR在本文中亦分別稱為「抗GPC3 caTCR」及「抗GPC3 CSR」)。應理解，當用於描述表現構築體或表現卡匣時，詞語「表現(express/expressing)」及「編碼(encode/encoding)」在本文中可互換使用。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.

在一些態樣中，本文提供一或多種表現構築體，其包含一或多個用於表現以下之表現卡匣：a)抗GPC3 caTCR，其包含：i)特異性結合GPC3的抗原結合模組；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該抗GPC3 CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，表現構築體為病毒載體。在一些實施例中，病毒載體係選自由以下組成之群：慢病毒載體、腺病毒載體、腺相關病毒載體、牛痘載體、單純疱疹病毒載體及EB病毒載體。在一些實施例中，表現構築體表現用於表現抗GPC3 caTCR、抗GPC3 CSR及c-Jun之多順反子表現卡匣。在一些實施例中，抗GPC3 caTCR之TCRM源於人類γ/δ TCR。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，抗GPC3 CSR之跨膜模組包含源於CD30 (例如人類CD30)之跨膜域。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組源於CD30 (例如人類CD30)。在一些實施例中，人類c-Jun多肽為野生型人類c-Jun多肽。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.

在一些實施例中，本文提供一或多種表現構築體，其包含一或多個用於表現以下之表現卡匣：a)抗GPC3 caTCR，其包含：i)抗原結合模組，該抗原結合模組包含：(1)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及(2)包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)配位體結合模組，該配位體結合模組包含：(1)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及(2)包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；ii)跨膜模組；及iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，抗GPC3 caTCR之抗原結合模組包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，caTCR為雜二聚體，其包含有包含SEQ ID NO: 30之胺基酸序列的第一多肽鏈及包含SEQ ID NO: 31之胺基酸序列的第二多肽鏈。在一些實施例中，caTCR為雜二聚體，其包含有包含SEQ ID NO: 10之胺基酸序列的第一多肽鏈及包含SEQ ID NO: 5之胺基酸序列的第二多肽鏈。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，抗GPC3 CSR之配位體結合模組包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR之抗GPC3配位體結合模組包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，抗GPC3 CSR之跨膜模組包含源於CD30 (例如人類CD30)之跨膜域。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組源於CD30 (例如人類CD30)。在一些實施例中，人類c-Jun多肽為野生型人類c-Jun。在一些實施例中，人類c-Jun多肽包含SEQ ID NO: 1之胺基酸序列。 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.

在一些實施例中，本文提供一或多種表現構築體，其包含一或多個用於表現以下之表現卡匣：a)抗GPC3 caTCR，其包含：i)包含特異性結合GPC3之Fab的抗原結合模組；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)包含能夠與GPC3結合或相互作用之scFv的配位體結合模組；ii)源於CD30之跨膜模組；及iii)源於CD30的能夠向免疫細胞提供協同刺激信號的協同刺激免疫細胞信號傳導模組，其中該抗GPC3 CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，Fab包含有包含SEQ ID NO: 28之胺基酸序列的重鏈可變域(V H)及包含SEQ ID NO: 29之胺基酸序列的輕鏈可變域(V L)。在一些實施例中，該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，該scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，抗GPC3 CSR包含CD30之片段。在一些實施例中，CD30之片段包含SEQ ID NO: 44之胺基酸序列。在一些實施例中，抗GPC3 CSR包含SEQ ID NO: 16之胺基酸序列。在一些實施例中，人類c-Jun多肽包含SEQ ID NO: 1之胺基酸序列。 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.

在其他態樣中，本文提供一種多順反子表現構築體，其包含用於表現以下之表現卡匣：a)抗GPC3 caTCR，其包含：i)特異性結合磷脂醯肌醇蛋白聚糖3 (GPC3)的抗原結合模組；及ii)衍生自人類γ/δ TCR之TCR模組(TCRM)；b)抗GPC3 CSR，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)衍生自人類CD30之胞內域的協同刺激免疫細胞信號傳導模組；及c)人類c-Jun多肽。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 30及31之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 10及5之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 30及31之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 10及5之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 30及31之編碼序列及SEQ ID NO: 16之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 10及5之編碼序列及SEQ ID NO: 16之編碼序列。在一些實施例中，表現卡匣進一步包含信號肽之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 2之編碼序列。在一些實施例中，編碼序列在框內藉由2A編碼序列或藉由內部核糖體進入位點(IRES)分開。在一些實施例中，表現卡匣包含組成性或可誘導型啟動子。在一些實施例中，啟動子為EF-1α啟動子。在一些實施例中，構築體為病毒載體(諸如慢病毒載體)。 A. 嵌合抗體 T 細胞受體 (caTCR) 構築體 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

本文提供之表現構築體編碼包含特異性結合GPC3之抗原結合模組的嵌合抗體T細胞受體(caTCR)構築體(在本文中亦被稱為「抗GPC3 caTCR」)。例示性caTCR提供於PCT/US2016/058305中，其內容以全文引用之方式併入本文中。抗GPC3 caTCR可以特異性結合GPC3且能夠募集至少一種TCR相關信號傳導分子(諸如CD3δε、CD3γε及/或ζζ)。在一些實施例中，抗GPC3 caTCR包含特異性結合細胞表面所結合之GPC3的抗原結合模組。在一些實施例中，提供一種抗GPC3 caTCR，其包含a)特異性結合GPC3的抗原結合模組；及b)包含第一TCR域(TCRD)及第二TCRD的TCR模組(TCRM)，該第一TCRD包含源於天然存在之TCR (諸如αβTCR或γδTCR)之跨膜域中之一者的第一TCR跨膜域(TCR-TM)，該第二TCRD包含源於天然存在之TCR (諸如αβTCR或γδTCR)之另一跨膜域的第二TCR-TM，其中該TCRM促進至少一種TCR相關信號傳導分子(諸如CD3δε、CD3γε及/或ζζ)的募集，且其中該抗原結合模組連接於第一及/或第二TCRD。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。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.

本文所述之抗GPC3 caTCR可以具有一或多個此部分中所述之特徵。意欲使本文所述之抗GPC3 caTCR之各組分(例如抗原結合模組、TCRD、TCR-TM、間隔模組、穩定模組、T細胞協同刺激序列、各種連接子等)的特徵中之任一者可彼此組合、與抗GPC3 CSR之特徵中之任一者及與c-Jun組合，如同每一組合經個別地描述一般。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.

在一些實施例中，抗原結合模組(諸如抗體部分)可特異性結合GPC3，其a)親和力為其對其他分子之結合親和力的至少約10 (包括例如至少約10、20、30、40、50、75、100、200、300、400、500、750、1000或更大中之任一者)倍；或b) K d不超過其結合其他分子之K d的約1/10 (諸如不超過約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或更小中之任一者)倍。結合親和力可以藉由此項技術中已知之方法測定，諸如ELISA、螢光活化細胞分選(FACS)分析或放射免疫沈澱分析(RIA)。K d可藉由此項技術中已知之方法，諸如利用例如Biacore儀器之表面電漿子共振(SPR)，或利用例如Sapidyne儀器之動力學排除分析(KinExA)測定。 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.

在一些實施例中，穩定模組包含第一穩定域及第二穩定域。在一些實施例中，第一及第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，穩定模組位於抗原結合模組與TCRM之間。在一些實施例中，穩定模組源於抗體部分。在一些實施例中，穩定模組源於抗體恆定域。在一些實施例中，穩定模組源於抗體重鏈恆定域。在一些實施例中，穩定模組中所含之抗體重鏈恆定域源於IgG (例如IgG1、IgG2、IgG3或IgG4)、IgA (例如IgA1或IgA2)、IgD、IgM或IgE重鏈，視情況為人類的。在一些實施例中，穩定模組中所含之抗體重鏈恆定域為相比於衍生其之序列包含一或多個修飾(例如，胺基酸取代、***及/或缺失)的變異體。在一些實施例中，穩定模組中所含之抗體輕鏈恆定域源於κ或λ輕鏈，視情況為人類的。在一些實施例中，穩定模組源於抗體輕鏈恆定域。在一些實施例中，穩定模組中所含之抗體輕鏈恆定域為相比於衍生其之序列包含一或多個修飾(例如，胺基酸取代、***及/或缺失)的變異體。在一些實施例中，第一及/或第二穩定域包含一或多個實質上不改變其彼此之間的結合親和力的修飾。在一些實施例中，第一及/或第二穩定域包含一或多個增加其彼此之間的結合親和力及/或引入非天然存在之二硫鍵的修飾。在一些實施例中，穩定模組包含杵臼修飾(knob-into-hole modification) (參見例如Carter P. J Immunol Methods.248:7-15, 2001)。舉例而言，在一些實施例中，穩定模組包含有包含杵臼修飾之抗體恆定域區域(例如C H3域)。在一些實施例中，穩定模組包含經靜電轉向修飾以增強其結合的抗體恆定域區域(參見例如WO2006106905 and Gunasekaran K等人. J Biol Chem.285:19637-46, 2010)。在一些實施例中，第一及第二穩定域藉由二硫鍵連接。 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.

穩定域的實例包括Fc區；鉸鏈區；C H2域；C H3域；C H4域；C H1域或C L域；TCR恆定域；白胺酸拉鏈域(例如，jun/fos白胺酸拉鏈域，參見例如Kostelney等人, J. Immunol, 148: 1547-1553, 1992；或酵母菌GCN4白胺酸拉鏈域)；異白胺酸拉鏈域；二聚化細胞表面受體之二聚化區(例如介白素-8受體(IL-8R)；或整合素雜二聚體，諸如LFA-1或GPIIIb/IIIa)；分泌的二聚化配位體之二聚化區(例如神經生長因子(NGF)、神經滋養素-3 (NT-3)、介白素-8 (IL-8)、血管內皮生長因子(VEGF)或大腦衍生神經滋養因子(BDNF)；參見例如Arakawa等人, J. Biol. Chem.269:27833-27839, 1994，及Radziejewski等人, Biochem. 32: 1350, 1993)；捲曲螺旋二聚化域(coiled coil dimerization domain) (參見例如WO2014152878; Fletcher等人, ACS Synth. Biol.1:240-250, 2012；及Thomas等人, J. Am. Chem. Soc.135(13):5161-5166, 2013)；及包含至少一個半胱胺酸殘基之多肽(例如約一個、兩個或三個至約十個半胱胺酸殘基)，使得可在該多肽及包含至少一個半胱胺酸殘基之第二多肽之間形式(一或多個)二硫鍵。 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.

在一些實施例中，本文所描述之TCRM包含第一T細胞受體域(TCRD)及第二TCRD，該第一TCRD包含第一TCR跨膜域(TCR-TM)，該第二TCRD包含第二TCR-TM，其中TCRM促進至少一種TCR相關信號傳導分子的募集。在一些實施例中，兩個TCR-TM均為天然存在的。在一些實施例中，該等TCR-TM中之至少一者為非天然存在的。在一些實施例中，兩個TCR-TM均為非天然存在的。在一些實施例中，第一TCR-TM源於T細胞受體(諸如αβ TCR或γδ TCR)之跨膜域中之一者，且第二TCR-TM源於T細胞受體之另一跨膜域。在一些實施例中，該TCRM相比於包含T細胞受體跨膜域之TCRM，增強至少一種TCR相關信號傳導分子之募集。TCR相關信號傳導分子之募集可藉由此項技術中已知之方法測定，該等方法諸如針對TCR-CD3複合物表面表現之FACS分析或CD3次單元與caTCR共免疫沈澱。舉例而言，在一些實施例中，本文所述之TCRM之第一TCR-TM包含TCR α鏈之跨膜域(例如GenBank登錄號：CCI73895)或其變異體、基本上由其組成或由其組成，且TCRM之第二TCR-TM包含TCR β鏈之跨膜域(例如GenBank登錄號：CCI73893)或其變異體、基本上由其組成或由其組成。在一些實施例中，第一TCR-TM包含TCR δ鏈之跨膜域(例如GenBank登錄號：AAQ57272)或其變異體、基本上由其組成或由其組成，且第二TCR-TM包含TCR γ鏈之跨膜域(例如GenBank登錄號：AGE91788)或其變異體、基本上由其組成或由其組成。在一些實施例中，本文所述之TCRM之第一及第二TCR-TM分別包含以下、基本上由以下組成或由以下組成：TCR α鏈恆定域之跨膜域(例如SEQ ID NO: 35)或其變異體及TCR β鏈恆定域之跨膜域(例如SEQ ID NO: 36)或其變異體。在一些實施例中，第一及第二TCR-TM分別包含以下、基本上由以下組成或由以下組成：TCR δ鏈恆定域之跨膜域(例如，SEQ ID NO: 37)或其變異體及TCR γ鏈恆定域之跨膜域(例如，SEQ ID NO: 38)或其變異體。在一些實施例中，第一及第二TCR-TM分別包含SEQ ID NO: 39及40之胺基酸序列或其變異體、基本上由其組成或由其組成。在一些實施例中，第一及第二TCR-TM分別包含SEQ ID NO: 41及42之胺基酸序列或其變異體、基本上由其組成或由其組成。跨膜域之變異體包括但不限於相比於參考序列具有一或多個胺基酸取代之跨膜域。在一些實施例中，相比於參考序列，變異跨膜域包含不超過5個胺基酸取代。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.

在一些實施例中，第一TCRD進一步包含在跨膜域之胺基端的第一連接肽及/或第二TCRD進一步包含在跨膜域之胺基端的第二連接肽。在一些實施例中，第一連接肽包含第一TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體，及/或第二連接肽包含第二TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體。在一些實施例中，第一及/或第二連接肽分別包含以下、基本上由以下組成或由以下組成：TCR α鏈恆定域之連接肽的全部或一部分或其變異體及TCR β鏈恆定域之連接肽的全部或一部分或其變異體。在一些實施例中，第一及/或第二連接肽分別包含以下、基本上由以下組成或由以下組成：TCR δ鏈恆定域之連接肽的全部或一部分或其變異體及TCR γ鏈恆定域之連接肽的全部或一部分或其變異體。在一些實施例中，第一TCRD進一步包含在第一TCR-TM之羧基端的第一TCR胞內域及/或第二TCRD進一步包含在第二TCR-TM之羧基端的第二TCR胞內域。在一些實施例中，第一TCR胞內域包含第一TCR-TM所源自之TCR次單元的胞內域之全部或一部分或其變異體，及/或第二TCR胞內域包含第二TCR-TM所源自之TCR次單元的胞內域之全部或一部分或其變異體。在一些實施例中，第一TCRD為天然存在之TCR之一條鏈的片段或其變異體，及/或第二TCRD為天然存在之TCR之另一條鏈的片段或其變異體。在一些實施例中，TCRD中之至少一者為非天然存在的。非天然存在之TCR域可為天然存在之TCR之對應域，其藉由一或多個胺基酸之取代及/或藉由用來自另一TCR之類似域的一部分置換對應域之一部分而經修飾。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.

在一些實施例中，第一TCR-TM包含以下、基本上由以下組成或由以下組成：SEQ ID NO: 3或與SEQ ID NO: 3包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列之全部或一部分。在一些實施例中，第二TCR-TM包含以下、基本上由以下組成或由以下組成：SEQ ID NO: 4或與SEQ ID NO: 4包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列之全部或一部分。在一些實施例中，第一及第二TCRD藉由二硫鍵連接。在一些實施例中，第一及第二TCRD藉由第一連接肽中之殘基與第二連接肽中之殘基之間的二硫鍵連接。在一些實施例中，TCRM能夠募集至少一種選自由CD3δε、CD3γε及ζζ組成之群的TCR相關信號傳導分子。在一些實施例中，TCRM能夠募集CD3δε、CD3γε及ζζ中之各者以形成caTCR-CD3複合物(亦即促進caTCR-CD3複合物形成)。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).

在一些實施例中，抗原結合模組為選自由以下組成之群的抗體部分：全長抗體、Fab、Fab'、(Fab') 2、Fv或單鏈Fv (scFv)。在一些實施例中，抗原結合模組為Fab或Fab'抗體部分。在一些實施例中，抗原結合模組為scFv抗體部分。在一些實施例中，抗體部分為全人源抗體部分、具有人類抗體構架區之半合成抗體部分或人源化抗體部分。 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.

在一些實施例中，抗原結合模組包含有包含抗GPC3 V H抗體域的第一抗原結合域及包含抗GPC3 V L抗體域的第二抗原結合域。在一些實施例中，抗GPC3 V H抗體域及抗GPC3 V L抗體域CDR源於同一抗體部分。在一些實施例中，抗GPC3 V H抗體域及抗GPC3 V L抗體域CDR中之一些源於不同抗體部分。在一些實施例中，抗GPC3 V H抗體域及/或抗GPC3 V L抗體域為人類抗體域、人源化抗體域、嵌合抗體域、半合成抗體域或完全合成抗體域。 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.

在一些實施例中，抗原結合模組為包含特異性CDR序列之抗體部分，該等特異性CDR序列源於一或多個對GPC3具有特異性之抗體部分(諸如單株抗體)或包含一或多個胺基酸取代的此類序列之某些變異體。在一些實施例中，變異序列中之胺基酸取代實質上並不降低抗原結合模組結合GPC3之能力。亦涵蓋實質上改善目標抗原結合親和力或影響一些其他特性(諸如對GPC3之相關變異體的特異性及/或與其之交叉反應性)的改變。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.

在一些實施例中，抗GPC3 caTCR包含連接於本文所述之TCRM的本文所述之抗原結合模組，視情況包括穩定模組。舉例而言，在一些實施例中，抗GPC3 caTCR包含連接於TCRD中之一或兩者之N端的抗原結合模組。在一些實施例中，抗GPC3 caTCR包含在TCRM與抗原結合模組之間的穩定模組。在一些實施例中，抗GPC3 caTCR進一步包含在任何兩個抗GPC3 caTCR模組或域之間的間隔模組。在一些實施例中，間隔模組包含長度在約5至約70個(諸如約5、10、15、20、25、30、35、40、45、50、55、60、65或70個中之任一者，包括此等值之間的任何範圍)胺基酸之間的一或多個肽連接子。在一些實施例中，抗GPC3 caTCR進一步包含一或多個輔助胞內域。在一些實施例中，一或多個輔助胞內域位於第一TCRD及/或第二TCRD之羧基端。在一些實施例中，一或多個輔助胞內域位於第一TCR-TM與第一TCR胞內域之間及/或第二TCR-TM與第二TCR胞內域之間。在一些實施例中，一或多個輔助胞內域個別地包含TCR協同刺激域。在一些實施例中，TCR協同刺激域包含免疫協同刺激分子(諸如CD27、CD28、4-1BB (CD137)、OX40、CD30、CD40、ICOS、淋巴細胞功能相關抗原-1 (LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、與CD83特異性結合之配位體及其類似物)之胞內域的全部或一部分。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).

在一些實施例中，本文所述之抗GPC3 caTCR包含a)特異性結合GPC3的抗原結合模組，及b)包含源於TCR (諸如αβTCR或γδTCR)之跨膜域的第一及第二TCR-TM的TCRM，其中TCRM能夠募集至少一種TCR相關信號傳導分子。在一些實施例中，抗原結合模組連接於TCRM中之一或多個多肽鏈之胺基端。舉例而言，在一些實施例中，TCRM包含兩個多肽鏈，且抗原結合模組連接於TCRM多肽鏈中之一或兩者之胺基端。在一些實施例中，第一及第二TCR-TM為天然存在的。在一些實施例中，該等TCR-TM中之至少一者為非天然存在的。在一些實施例中，第一及第二TCR-TM為非天然存在的。在一些實施例中，TCRM進一步包含在TCR-TM之胺基端的TCR之至少一個連接肽或其片段。在一些實施例中，TCRM進一步包含至少一個TCR胞內域，該至少一個TCR胞內域包含來自在TCR-TM之羧基端的TCR胞內域的序列。在一些實施例中，TCRM包含源於TCR鏈之片段的TCRD。在一些實施例中，TCRD中之至少一者為非天然存在的。在一些實施例中，caTCR進一步包含在TCR-TM之羧基端的包含T細胞協同刺激信號傳導序列(諸如來自CD27、CD28、4-1BB (CD137)、OX40、CD30或CD40)的至少一個輔助胞內域。在一些實施例中，caTCR缺乏協同刺激信號傳導序列。在一些實施例中，抗原結合模組為特異性結合GPC3的抗體部分(「抗GPC3抗體部分」)。在一些實施例中，抗GPC3抗體部分包含抗GPC3 V H抗體域及抗GPC3 V L抗體域。在一些實施例中，抗GPC3抗體部分為人類抗體域、人源化抗體域、嵌合抗體域、半合成抗體域或完全合成抗體域。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，穩定模組包含至少一個連接穩定域之二硫鍵。在一些實施例中，第一及第二穩定域包含抗體域(諸如C H1及C L抗體域)或其變異體。在一些實施例中，TCRM能夠募集至少一種選自由CD3δε、CD3γε及ζζ組成之群的TCR相關信號傳導分子。在一些實施例中，TCRM相較於包含T細胞受體跨膜域之TCRM，允許至少一種TCR相關信號傳導分子之募集增強。在一些實施例中，TCRM促進caTCR-CD3複合體形成。在一些實施例中，任何兩個抗GPC3 caTCR模組或域之間存在間隔模組。在一些實施例中，抗GPC3 caTCR為雜多聚體，諸如雜二聚體。舉例而言，在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含有包含第一TCRD的第一多肽鏈及包含第二TCRD的第二多肽鏈，其中該抗原結合模組連接於第一及/或第二多肽鏈。 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.

在一些實施例中，本文所述之抗GPC3 caTCR特異性結合GPC3，且包含：a)第一TCRD，其包含源於TCR跨膜域中之一者的第一TCR-TM；及第二TCRD，其包含源於TCR之另一跨膜域的第二TCR-TM，其中第一及第二TCRD形成能夠募集至少一種TCR相關信號傳導分子的TCRM；及b)特異性結合GPC3的抗原結合模組，其中抗原結合模組連接於第一及/或第二TCRD。在一些實施例中，兩個TCR-TM均為天然存在的。在一些實施例中，該等TCR-TM中之至少一者為非天然存在的。在一些實施例中，TCR為αβ TCR且第一及第二TCR-TM源於TCR α及β次單位跨膜域。在一些實施例中，TCR為γδ TCR，且第一及第二TCR-TM源於TCR γ及δ次單位跨膜域。在一些實施例中，第一TCRD進一步包含第一TCR連接肽或其片段，及/或第二TCRD進一步包含第二TCR連接肽或其片段。在一些實施例中，第一連接肽包含第一TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體，及/或第二連接肽包含第二TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體。在一些實施例中，第一及第二連接肽藉由二硫鍵連接。在一些實施例中，第一TCRD進一步包含第一TCR胞內域，及/或第二TCRD進一步包含第二TCR胞內域。在一些實施例中，第一TCR胞內域包含來自第一TCR-TM所源自之TCR次單元的胞內域的序列，及/或第二TCR胞內域包含來自第二TCR-TM所源自之TCR次單元的胞內域的序列。在一些實施例中，第一TCRD為第一TCR-TM所源自之TCR次單元的片段，及/或第二TCRD為第二TCR-TM所源自之TCR次單元的片段。在一些實施例中，抗GPC3 caTCR進一步包含至少一個包含T細胞協同刺激信號傳導序列(諸如來自CD27、CD28、4-1BB (CD137)、OX40、CD30或CD40)的輔助胞內域。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，第一及第二穩定域藉由二硫鍵連接。在一些實施例中，第一及第二穩定域包含抗體域(諸如C H1及C L抗體域)或其變異體。在一些實施例中，TCRM能夠募集至少一種選自由CD3δε、CD3γε及ζζ組成之群的TCR相關信號傳導分子。在一些實施例中，TCRM相較於包含T細胞受體跨膜域之TCRM，允許至少一種TCR相關信號傳導分子之募集增強。在一些實施例中，TCRM促進caTCR-CD3複合體形成。在一些實施例中，任何兩個抗GPC3 caTCR模組或域之間存在間隔模組。在一些實施例中，抗原結合模組為抗體部分(例如及抗GPC3抗體部分)。在一些實施例中，抗體部分為Fab、Fab'、(Fab') 2、Fv或單鏈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 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).

在一些實施例中，本文所述之抗GPC3 caTCR特異性結合GPC3，且包含：a)第一TCRD，其包含源於天然存在之γδ TCR之跨膜域中之一者的第一TCR-TM；及第二TCRD，其包含源於天然存在之γδ TCR之另一跨膜域的第二TCR-TM，其中第一及第二TCRD形成能夠募集至少一種TCR相關信號傳導分子的TCRM；及b)特異性結合GPC3a的抗原結合模組，其中抗原結合模組連接於第一及/或第二TCRD。在一些實施例中，兩個TCR-TM均為天然存在的。在一些實施例中，該等TCR-TM中之至少一者為非天然存在的。在一些實施例中，第一TCRD進一步包含第一TCR連接肽或其片段，及/或第二TCRD進一步包含第二TCR連接肽或其片段。在一些實施例中，第一連接肽包含第一TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體，及/或第二連接肽包含第二TCR-TM所源自之TCR次單元的連接肽之全部或一部分或其變異體。在一些實施例中，第一及第二連接肽藉由二硫鍵連接。在一些實施例中，第一TCRD進一步包含第一TCR胞內域，及/或第二TCRD進一步包含第二TCR胞內域。在一些實施例中，第一TCR胞內域包含來自第一TCR-TM所源自之TCR次單元的胞內域的序列，及/或第二TCR胞內域包含來自第二TCR-TM所源自之TCR次單元的胞內域的序列。在一些實施例中，第一TCRD為第一TCR-TM所源自之TCR次單元的片段，及/或第二TCRD為第二TCR-TM所源自之TCR次單元的片段。在一些實施例中，抗GPC3 caTCR進一步包含至少一個包含T細胞協同刺激信號傳導序列(諸如來自CD27、CD28、4-1BB (CD137)、OX40、CD30或CD40)的輔助胞內域。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，第一及第二穩定域藉由二硫鍵連接。在一些實施例中，第一及第二穩定域包含抗體域(諸如C H1及C L抗體域)或其變異體。在一些實施例中，TCRM能夠募集至少一種選自由CD3δε、CD3γε及ζζ組成之群的TCR相關信號傳導分子。在一些實施例中，TCRM相比於包含天然存在之γδ T細胞受體跨膜域的TCRM，允許至少一種TCR相關信號傳導分子之募集增強。在一些實施例中，TCRM促進caTCR-CD3複合體形成。在一些實施例中，任何兩個抗GPC3 caTCR模組或域之間存在間隔模組。在一些實施例中，抗原結合模組為抗體部分(例如及抗GPC3抗體部分)。在一些實施例中，抗體部分為Fab、Fab'、(Fab') 2、Fv或單鏈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).

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含GDN之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L。在一些實施例中，抗原結合模組為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: 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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含YDS之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L。在一些實施例中，抗原結合模組為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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含DDS之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。在一些實施例中，抗GPC3抗體部分為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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24包含具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：a)包含SEQ ID NO: 24之胺基酸序列的V H；及b)包含SEQ ID NO: 25之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 24之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 25之胺基酸序列之V L的LC-CDR。在一些實施例中，抗原結合模組為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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：a)包含SEQ ID NO: 26之胺基酸序列的V H；及b)包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 26之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 27之胺基酸序列之V L的LC-CDR。在一些實施例中，抗原結合模組為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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：a)包含SEQ ID NO: 28之胺基酸序列的V H；及b)包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 28之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 29之胺基酸序列之V L的LC-CDR。在一些實施例中，抗原結合模組為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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含有包含第一TCRD的第一多肽鏈及包含第二TCRD的第二多肽鏈。在一些實施例中，抗GPC3 caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與源於TCR α鏈之TCRD融合的V H，該第二多肽鏈包含與源於TCR β鏈之TCRD融合的V L。在一些實施例中，抗GPC3 caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與源於TCR β鏈之TCRD融合的V H，該第二多肽鏈包含與源於TCR α鏈之TCRD融合的V L。在一些實施例中，抗GPC3 caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V L。在一些實施例中，抗GPC3 caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V L。如本文所用，術語「融合」意謂直接融合或間接融合。 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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 30之胺基酸序列或與SEQ ID NO: 30具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 31之胺基酸序列或與SEQ ID NO: 31具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 30之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 31之胺基酸序列的第二多肽鏈。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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 10之胺基酸序列或與SEQ ID NO: 10具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 5之胺基酸序列或與SEQ ID NO: 5具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 10之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 5之胺基酸序列的第二多肽鏈。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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 63之胺基酸序列或與SEQ ID NO: 63具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 64之胺基酸序列或與SEQ ID NO: 64具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 63之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 64之胺基酸序列的第二多肽鏈。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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 65之胺基酸序列或與SEQ ID NO: 65具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 66之胺基酸序列或與SEQ ID NO: 66具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 65之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 66之胺基酸序列的第二多肽鏈。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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 67之胺基酸序列或與SEQ ID NO: 67具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 68之胺基酸序列或與SEQ ID NO: 68具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 67之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 68之胺基酸序列的第二多肽鏈。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.

在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)第一多肽鏈，其包含SEQ ID NO: 69之胺基酸序列或與SEQ ID NO: 69具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列；及ii)第二多肽鏈，其包含SEQ ID NO: 70之胺基酸序列或與SEQ ID NO: 70具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性的胺基酸序列。在一些實施例中，抗GPC3 caTCR為雜二聚體，其包含：i)包含SEQ ID NO: 69之胺基酸序列的第一多肽鏈，及ii)及包含SEQ ID NO: 70之胺基酸序列的第二多肽鏈。 B. 嵌合協同刺激受體 (CSR) 構築體 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

本文提供之表現構築體進一步編碼特異性結合GPC3 (在本文中亦被稱為「抗GPC3 CSR」)的嵌合協同刺激受體(CSR)。抗GPC3 CSR能夠在目標配位體結合之後刺激表面上的功能性表現該CSR的免疫細胞。抗GPC3 CSR包含提供配位體結合特異性的配位體結合模組、跨膜模組及允許刺激免疫細胞的協同刺激免疫細胞信號傳導模組。抗GPC3 CSR缺乏功能性初級免疫細胞信號傳導序列。在一些實施例中，抗GPC3 CSR缺乏任何初級免疫細胞信號傳導序列。在一些實施例中，抗GPC3 CSR包含有包含配位體結合模組、跨膜模組及協同刺激信號傳導模組的單一多肽鏈。在一些實施例中，抗GPC3 CSR包含第一多肽鏈及第二多肽鏈，其中第一與第二多肽鏈一起形成配位體結合模組、跨膜模組及協同刺激信號傳導模組。在一些實施例中，第一及第二多肽鏈為單獨的多肽鏈，且抗GPC3 CSR為多聚體，諸如二聚體。在一些實施例中，第一多肽鏈與第二多肽鏈共價連接，諸如藉由肽鍵或藉由另一種化學鍵(諸如二硫鍵)連接。在一些實施例中，第一多肽鏈與第二多肽鏈藉由至少一個二硫鍵連接。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.

本發明之抗GPC3 CSR中所用的協同刺激免疫細胞信號傳導域之實例包括：T細胞受體(TCR)之共受體的細胞質序列，該等細胞質序列可與caTCR (例如，如本文所述之抗GPC3)協同作用以在caTCR接合之後起始信號轉導；以及此等序列之任何衍生物或變異體及具有相同功能能力的任何合成序列。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.

在一些情形下，經由單獨的TCR產生之信號不足以完全活化T細胞，且亦需要協同刺激信號。因此，在一些實施例中，T細胞活化係由兩種相異類別之胞內信號傳導序列介導：經由TCR起始抗原依賴性初級活化之序列(在本文中稱為「初級T細胞信號傳導序列」)及以抗原非依賴性方式起作用以提供二級或協同刺激信號之序列(在本文中稱為「協同刺激T細胞信號傳導序列」)。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").

以刺激方式起作用之初級免疫細胞信號傳導序列可含有信號傳導模體，其稱為基於免疫受體酪胺酸之活化模體或ITAM。含ITAM之初級免疫細胞信號傳導序列之實例包括源於TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b及CD66d之彼等序列。「功能性」初級免疫細胞信號傳導序列為在與適當受體可操作地偶合時能夠轉導免疫細胞活化信號之序列。可包含初級免疫細胞信號傳導序列之片段或變異體之「非功能性」初級免疫細胞信號傳導序列不能轉導免疫細胞活化信號。本文所述之抗GPC3 CSR缺乏功能性初級免疫細胞信號傳導序列，諸如包含ITAM的功能性信號傳導序列。在一些實施例中，抗GPC3 CSR缺乏任何初級免疫細胞信號傳導序列。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.

協同刺激免疫細胞信號傳導序列可為協同刺激分子之胞內域的一部分，該協同刺激分子包括例如CD27、CD28、4-1BB (CD137)、OX40、CD27、CD40、PD-1、ICOS、淋巴細胞功能相關抗原-1 (LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、與CD83特異性結合的配位體，及其類似物。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.

抗GPC3 CSR之目標配位體為GPC3。在一些實施例中，GPC3為細胞表面結合之GPC3。在一些實施例中，該GPC3目標配位體與表現於同一免疫細胞中之caTCR (例如，如本文所述之抗GPC3 caTCR)的GPC3目標抗原相同。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.

在一些實施例中，抗GPC3 CSR中的配位體結合模組為特異性結合GPC3 的抗體部分(「抗GPC3抗體部分」)。在一些實施例中，抗體部分為Fab、Fab'、(Fab') 2、Fv或單鏈Fv (scFv)。在一些實施例中，配位體結合模組源於受體之胞外域。在一些實施例中，抗GPC3 CSR之抗體部分包含對GPC3具有特異性之抗體部分的CDR或可變域(V H及/或V L域)。 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.

在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含GDN之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L。在一些實施例中，配位體結合模組為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: 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.

在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含YDS之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L。在一些實施例中，配位體結合模組為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.

在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V H，以及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體、包含DDS之胺基酸序列的LC-CDR2或包含至多約3 (諸如約1、2或3中之任一者)個胺基酸取代之其變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或包含至多約5 (諸如約1、2、3、4或5中之任一者)個胺基酸取代之其變異體的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H，以及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。在一些實施例中，配位體結合模組為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.

在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24包含具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：a)包含SEQ ID NO: 24之胺基酸序列的V H；及b)包含SEQ ID NO: 25之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含SEQ ID NO: 24之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 25之胺基酸序列之V L的LC-CDR。在一些實施例中，配位體結合模組為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.

在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：a)包含SEQ ID NO: 26之胺基酸序列的V H；及b)包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含SEQ ID NO: 26之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 27之胺基酸序列之V L的LC-CDR。在一些實施例中，配位體結合模組為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.

在一些實施例中，抗GPC3 caTCR包含抗原結合模組，該抗原結合模組包含：包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29包含至少約具有至少約80% (包括例如至少約80%、85%、90%、95%、96%、97%、98%或99%中之任一者)序列一致性之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：a)包含SEQ ID NO: 28之胺基酸序列的V H；及b)包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR包含配位體結合模組，該配位體結合模組包含：包含SEQ ID NO: 28之胺基酸序列之V H的HC-CDR，及包含SEQ ID NO: 29之胺基酸序列之V L的LC-CDR。在一些實施例中，配位體結合模組為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.

在一些實施例中，抗GPC3 CSR之跨膜域包含源於跨膜蛋白之跨膜域，該跨膜蛋白包括例如CD28、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137或CD154。在一些實施例中，抗GPC3 CSR包含衍生自CD30之跨膜域的跨膜域。在一些實施例中，CD30跨膜域包含SEQ ID NO: 49之胺基酸序列或其包含與SEQ ID NO: 49具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。在一些實施例中，抗GPC3 CSR包含源於CD28之跨膜域的跨膜域。在一些實施例中，CD28跨膜域包含SEQ ID NO: 50之胺基酸序列或其包含與SEQ ID NO: 50具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。在一些實施例中，抗GPC3 CSR包含源於CD8跨膜域的跨膜域。在一些實施例中，CD8跨膜域包含SEQ ID NO: 22之胺基酸序列或其包含與SEQ ID NO: 22具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。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.

在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組衍生自TCR之協同刺激受體的胞內域。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組包含以下、基本上由以下組成或由以下組成：例如CD27、CD28、4-1BB (CD137)、OX40、CD30、CD40、ICOS、淋巴細胞功能相關抗原-1 (LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、與CD83特異性結合之配位體及其類似物的協同刺激受體之胞內域之全部或一部分。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組包含以下、基本上由以下組成或由以下組成：CD30、CD28、4-IBB、OX40、ICOS、CD27或CD40之協同刺激受體的胞內域的全部或一部分。在一些實施例中，CSR之協同刺激免疫細胞信號傳導模組衍生自人類CD30。在一些實施例中，源於人類CD30之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 44之胺基酸序列或其包含與SEQ ID NO: 44具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。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.

在一些實施例中，CSR之協同刺激免疫細胞信號傳導模組源於人類CD28。在一些實施例中，源於人類CD28之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 45之胺基酸序列或其包含與SEQ ID NO: 45具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。在一些實施例中，源於人類CD28之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 46之胺基酸序列或其包含與SEQ ID NO: 46具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。在一些實施例中，CSR之協同刺激免疫細胞信號傳導模組源於人類4-IBB。在一些實施例中，源於人類4-IBB之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 47之胺基酸序列或其包含與SEQ ID NO: 47具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。在一些實施例中，源於人類4-IBB之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 48之胺基酸序列或其包含與SEQ ID NO: 48具有至少約85%序列一致性(例如至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之胺基酸序列的功能等效物。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.

在一些實施例中，抗GPC3 CSR進一步包含在配位體結合模組、跨膜模組及協同刺激信號傳導模組中之任兩者之間的間隔模組。在一些實施例中，間隔模組包含連接兩個抗GPC3 CSR模組的一或多個肽連接子。在一些實施例中，間隔模組包含長度在約5至約70個(諸如約5、10、15、20、25、30、35、40、45、50、55、60、65或70個中之任一者，包括此等值之間的任何範圍)胺基酸之間的一或多個肽連接子。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.

在一些實施例中，抗GPC3 CSR之配位體結合模組(諸如抗體部分)特異性結合GPC3，其a)親和力可為其對其他分子之結合親和力的至少約10 (包括例如至少約10、20、30、40、50、75、100、200、300、400、500、750、1000或更大中之任一者)倍；或b) K d可超過其結合其他分子之K d的約1/10 (諸如不超過約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或更小中之任一者)倍。結合親和力可以藉由此項技術中已知之方法測定，諸如ELISA、螢光活化細胞分選(FACS)分析或放射免疫沈澱分析(RIA)。K d可藉由此項技術中已知之方法，諸如利用例如Biacore儀器之表面電漿子共振(SPR)，或利用例如Sapidyne儀器之動力學排除分析(KinExA)測定。 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.

在一些實施例中，本文所述之抗GPC3 CSR包含a) scFv；及b) CD30片段。在一些實施例中，scFv包含具有SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26具有至少約90%序列一致性(例如至少約91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之其變異體的V H域，及具有SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27具有至少約90%序列一致性(例如至少約91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之其變異體的V L域。在一些實施例中，scFv包含具有SEQ ID NO: 26之胺基酸序列的V H域及具有SEQ ID NO: 27之胺基酸序列的V L域。在一些實施例中，scFv包含以下、基本上由以下組成或由以下組成：SEQ ID NO: 33之胺基酸序列或與SEQ ID NO: 33具有至少約90%序列一致性(例如至少約91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之其變異體。在一些實施例中，scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，CD30之片段包含SEQ ID NO: 44之胺基酸序列。在一些實施例中，CSR自胺基端至羧基端包含scFv、肽連接子及CD30之片段。在一些實施例中，CSR包含SEQ ID NO: 44之胺基酸序列、基本上由該胺基酸序列組成或由該胺基酸序列組成。在一些實施例中，抗GPC3 CSR包含SEQ ID NO: 16之胺基酸序列或與SEQ ID NO: 16具有至少約90%序列一致性(例如至少約91%、92%、93%、94%、95%、96%、97%、98%或99%序列一致性)之其變異體。在一些實施例中，抗GPC3 CSR包含SEQ ID NO: 16之胺基酸序列。 C. c-Jun 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.

本文提供之表現構築體進一步編碼c-Jun多肽。The expression constructs provided herein further encode a c-Jun polypeptide.

在一些實施例中，c-Jun為人類c-Jun。在一些實施例中，人類c-Jun為野生型人類c-Jun。在一些實施例中，人類c-Jun具有以下胺基酸序列(在GenBank以登錄號AAA59197.1或在UniProtKB (以登錄號P05412.2)獲得： 亦參加Hattori等人, PNAS(1988) 85:9148-52。 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.

在人類中，c-Jun蛋白由位於染色體1 (GenBank登錄號NC_000001.11之核苷酸58,780,791至58,784,047，負股取向)上之JUN基因編碼。JUN基因及其編碼蛋白的同義詞已知且包括「Jun原癌基因，AP-1轉錄因子次單位」、「v-Jun禽類肉瘤病毒17致癌基因同源物」、「轉錄因子AP-1」、「Jun致癌基因」、「AP-1」、「Jun活化域結合蛋白」、「p39」及「強化子結合蛋白AP1」。野生型人類c-Jun蛋白質序列之長度為331個胺基酸。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.

在一些實施例中，野生型人類c-Jun與SEQ ID NO: 1之胺基酸序列具有至少約90% (例如至少約91%、92%、93%、94%、95%、96%、97%、98%或99%)一致性。在一些實施例中，野生型人類c-Jun包含SEQ ID NO: 1之胺基酸序列。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.

在一些實施例中，c-Jun為突變型c-Jun。在一些實施例中，c-Jun為突變型人類c-Jun。在一些實施例中，突變型c-Jun不影響突變體拯救功能異常(耗竭)之T細胞的能力。在一些實施例中，突變型c-Jun與野生型c-Jun之C端胺基酸殘基(例如，C端50、75、100、150、200、250個或更多個胺基酸殘基)、C端部分(例如，四分之一、三分之一或二分之一)或C端域(例如，其ε、bZIP及胺基酸C端)具有至少約70% (例如至少約75%、80%、85%、90%、95%或99%)序列一致性。在一些實施例中，野生型c-Jun之N端胺基酸殘基(例如，N端50、75、100或150個或更多)、N端部分(例如，四分之一、三分之一或二分之一)或N端域(例如，其δ、反式活化域及胺基酸N端)缺失、突變或以其他方式不活化。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.

在一些實施例中，c-Jun在其反式活化域中包含一或多個不活化突變(例如取代、缺失或***)。在一些實施例中，c-Jun在其反式活化域中包含一或多個不活化取代。在一些實施例中，c-Jun在其反式活化域中包含一或多個不活化缺失。在一些實施例中，c-Jun在其反式活化域中包含一或多個不活化***。在一些實施例中，c-Jun在其δ域中包含一或多個不活化突變(例如取代、缺失或***)。在一些實施例中，c-Jun在其δ域中包含一或多個不活化取代。在一些實施例中，c-Jun在其δ域中包含一或多個不活化缺失。在一些實施例中，c-Jun在其δ域中包含一或多個不活化***。在一些實施例中，c-Jun在其反式活化域及其δ域中包含一或多個不活化突變(例如取代、缺失或***)。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.

在一些實施例中，c-Jun包含絲胺酸(S)至丙胺酸(A)的取代。在一些實施例中，c-Jun包含相比於SEQ ID NO: 1之S63A及S73A取代中之一者或兩者(在上文人類c-Jun之胺基酸序列中框出了位置)。在一些實施例中，c-Jun具有相比於SEQ ID NO: 1之在胺基酸殘基2與胺基酸殘基102之間或殘基30與50之間的缺失。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.

在某些實施例中，抗GPC3 caTCR及/或抗GPC3 CSR及/或c-Jun多肽進一步包含可供用於各種用途的親和力或純化標籤；舉例而言，其可用於增強目標多肽的純化效率。在一個實施例中，標籤為myc、HIS或HA標籤。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.

在一些態樣中，本文所述之免疫細胞經修飾過以包含編碼c-Jun多肽之外源性核苷酸序列，其中該外源性核苷酸序列與SEQ ID NO: 52-62中所列之核酸序列中之任一者具有至少約50%、至少約55%、至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸包含SEQ ID NO: 52-62中之任一者中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 52中所列之核酸序列具有至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 52中所列之核酸序列具有至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 52中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 53中所列之核酸序列具有至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 53中所列之核酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 53中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 54中所列之核酸序列具有至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 54中所列之核酸序列具有至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 54中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 55中所列之核酸序列具有至少約50%、至少約55%、至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 55中所列之核酸序列具有至少96%、至少97%、至少98%或至少99%一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 55中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 56中所列之核酸序列具有至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 56中所列之核酸序列具有至少79%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 56中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 57中所列之核酸序列具有至少約80%、至少85%、至少90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 57中所列之核酸序列具有至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 57中所列之核酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 58中所列之核酸序列具有至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 58中所列之核酸序列具有至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 58中所列之核苷酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 59中所列之核酸序列具有至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 59中所列之核酸序列具有至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 59中所列之核苷酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 60中所列之核酸序列具有至少約55%、至少約60%、至少約65%、至少約70%、至少約75%、至少約80%、至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 60中所列之核酸序列具有至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性聚核苷酸包含SEQ ID NO: 60中所列之核苷酸序列。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.

在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 61中所列之核酸序列具有至少約85%、至少約90%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%序列一致性。在一些態樣中，編碼c-Jun多肽之外源性聚核苷酸與SEQ ID NO: 61中所列之核酸序列具有至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在一些態樣中，外源性核苷酸包含SEQ ID NO: 61中所列之核苷酸序列。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.

例示性c-Jun核苷酸序列提供於本文所提供之序列表中。Exemplary c-Jun nucleotide sequences are provided in the Sequence Listing provided herein.

可使用此項技術中已知之任何方法使本文所揭示之c-Jun核苷酸序列密碼子最佳化。舉例而言，在一些態樣中，本文所揭示之c-Jun核苷酸序列之密碼子已經最佳化以相對於野生型核苷酸序列(例如SEQ ID NO: 62)修改(例如增加或減少)以下參數中之一或多者：(i)密碼子適應指數((亦即密碼子使用偏好)；(ii)鳥嘌呤-胞嘧啶(GC)核苷酸含量；(iii) mRNA二級結構及不穩定模體；(iv)重複序列(例如正向重複序列、反向重複序列、二元重複序列(dyad repeat))；(v)限制酶識別位點；或(vi)其組合。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.

雖然本文提供的某些揭示內容大體上關於修飾免疫細胞以包含編碼c-Jun蛋白(野生型c-Jun或其變異體)的外源性核苷酸序列，但對於本領域技術人員而言將顯而易見的是，可使用其他適合的方法在細胞中誘導及/或增加c-Jun蛋白表現(野生型或其變異體)。舉例而言，如本文所描述，在一些態樣中，可用轉錄活化因子(例如，CRISPRa)增加內源性c-Jun蛋白表現。除非另外指示，否則使用外源性核苷酸序列之上文所提供之揭示內容同樣適用於誘導及/或增加本文所提供之細胞中之c-Jun蛋白表現的其他方法(例如轉錄活化因子，例如CRISPRa)。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.

在一些態樣中，歸因於修飾(例如，外源性引入之c-Jun核苷酸序列及/或轉錄活化因子之引入)，經工程改造之細胞過度表現，亦即表現比不含此類修飾之對應細胞(「參考細胞」)高的c-Jun蛋白質含量(例如，高至少約10%、20%、30%、40%、50%、60%、70%、80%、90%或100%，或高至少約1.5倍、2倍、3倍、4倍、5倍或10倍)。術語「表現增加之含量[或量]」、「過度表現」或具有「增加之表現」(及本文所用片語之類似形式)可互換使用。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.

在一些態樣中，本文所述之經工程改造(或經修飾)之細胞表現比參考細胞多至少約2-100倍、多約5-50倍、多約5-40倍、多約5-30倍、多約5-20倍、多約8-20倍或多10-20倍之c-Jun蛋白質。在一些態樣中，與參考細胞中之c-Jun蛋白質之表現相比，本文所述之經修飾細胞中的c-Jun蛋白質之表現增加至少約0.5倍、至少約1倍、至少約2倍、至少約3倍、至少約4倍、至少約5倍、至少約6倍、至少約7倍、至少約8倍、至少約9倍、至少約10倍、至少約12倍、至少約14倍、至少約16倍、至少約18倍、至少約20倍、至少約25倍、至少約30倍、至少約35倍、至少約40倍、至少約45倍或至少約50倍。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.

在一些態樣中，增加之c-Jun蛋白表現可改善及/或增強經修飾免疫細胞(例如，T細胞，諸如CD4+及/或CD8+ T細胞)之一或多個特性。此類特性之非限制性實例包括：耗竭抗性(例如，藉由耗竭標記物，諸如PD-1、CD39、TIM-3及/或LAG-3之減少之表現所指示；持久性/存活率增加；功能異常狀態之發作延遲；及/或細胞介素(例如IFN-γ及/或IL-2)產量增加)；增加之擴增/增殖；增加之抗原敏感性；改善之效應功能，尤其是在重複抗原刺激之後的改善之效應功能(例如在抗原刺激後產生細胞介素，表現目標抗原之細胞溶解，或此二者)；或其組合。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.

適用於量測耗竭、細胞表型、持久性、細胞毒性及/或殺傷、增殖、細胞介素產生/釋放及基因表現概況之分析為此項技術中已知的，且包括例如流動式細胞測量術、細胞內細胞介素染色(ICS)、INCUCYTE®免疫細胞殺傷分析、Meso Scale Discovery (MSD，一個將電致化學發光與MULTI-ARRAY技術組合之平台)或類似分析、持久性抗原刺激分析、大量及單個細胞RNAseq (參見例如Front Genet. 2020; 11:220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci. 2:139-173)、細胞毒性/殺傷分析、ELISA、西方墨點及其他標準分子及細胞生物學方法(諸如本文所描述的或例如在Current Protocols in Molecular Biology or Current Protocols in Immunology (John Wiley & Sons, Inc., 1999-2021)中所描述的)或其他分析。 D. 核酸 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

抗GPC3 caTCR、抗GPC3 CSR及c-Jun可經由一或多個核酸分子(例如DNA或RNA，諸如mRNA)引入T細胞或祖細胞。在一些實施例中，核酸分子可置於一或多個DNA或RNA載體上以引入宿主細胞中。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.

可藉由熟知技術將核酸分子(例如含有其之DNA或RNA載體)引入細胞中，包括但不限於電穿孔、磷酸鈣沈澱、脂質體轉染、粒子轟擊、顯微注射、膠態分散系統(例如，作為大分子複合物、奈米膠囊、微球體及珠粒)及基於脂質之系統(例如，水包油乳液、微胞、混合微胞及脂質體)。替代地，可藉由轉導重組病毒將核酸分子引入細胞中，該等重組病毒之基因體包含核酸分子。病毒載體之實例包括但不限於源於慢病毒、反轉錄病毒、腺病毒、腺相關病毒、單純疱疹病毒、仙台病毒(Sendai virus)及牛痘病毒之載體。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.

在一些實施例中，抗GPC3 caTCR、抗GPC3 CSR及c-Jun之兩條多肽鏈的編碼序列可置於單一表現構築體上。四個編碼序列可置於構築體上之一或多個表現卡匣中，各卡匣為其自身的轉錄單元(例如用其自身的啟動子及聚腺苷酸化位點及其他轉錄控制元件)。在特定實施例中，可將四個編碼序列置於單一表現卡匣(例如多順反子表現卡匣)中，其中四個編碼序列在共同啟動子下轉錄。在多順反子排列中，編碼序列為同框的且藉由自裂解肽(例如2A自裂解肽，諸如T2A、P2A、E2A或F2A肽)之編碼序列彼此分開。替代地，編碼序列可藉由核糖體內部進入位點(IRES)彼此分開。因此，多順反子(例如四順反子)表現卡匣轉錄成單一RNA，但最終單一RNA經處理且轉譯成獨立多肽。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.

在一些實施例中，多順反子表現卡匣包含抗GPC3 caTCR、抗GPC3 CSR及c-Jun之編碼序列。在一些實施例中，表現卡匣包含抗GPC3 caTCR之抗原結合模組的編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 28及29之胺基酸序列的編碼序列。在一些實施例中，表現卡匣包含衍生自人類γ/δ TCR之TCRM之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 4及5之胺基酸序列的編碼序列。在一些實施例中，表現卡匣包含抗GPC3 caTCR之兩條多肽鏈的編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 30及31之胺基酸序列的編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 10及5之胺基酸序列的編碼序列。在一些實施例中，表現卡匣包含抗GPC3 CSR之配位體結合模組的編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 33之胺基酸序列之編碼序列。在一些實施例中，表現卡匣包含抗GPC3 CSR之人類CD30之部分序列的編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 43之胺基酸序列的編碼序列。在一些實施例中，表現卡匣包含人類c-Jun多肽之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 30及31之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 10及5之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 30及31之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 10及5之編碼序列及SEQ ID NO: 33及43之編碼序列。在一些實施例中，表現卡匣進一步包含信號肽之編碼序列。在一些實施例中，表現卡匣包含SEQ ID NO: 2之編碼序列。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.

表現卡匣(多順反子或單順反子)可包含在哺乳動物(例如人類或人類T)細胞中具有組成型活性之啟動子。此類啟動子包括但不限於即刻早期細胞巨大病毒(CMV)啟動子、猿猴病毒40 (SV40)早期啟動子、人類免疫缺乏病毒(HIV)長末端重複序列(LTR)啟動子、艾司坦-巴爾病毒即刻早期啟動子、勞斯肉瘤病毒(Rous sarcoma virus)啟動子、延伸因子-1α (EF-1α)啟動子、MND啟動子、肌動蛋白啟動子、肌凝蛋白啟動子、血紅蛋白啟動子及肌酸激酶啟動子。亦涵蓋源於前述啟動子之核心或最小啟動子。替代地，表現卡匣可包含誘導型啟動子系統。例示性誘導型啟動子系統包括但不限於激素調節元件、合成性配位體調節元件、電離輻射調節元件、四環素(Tet)系統(例如「Tet-Off」及「Tet-On」系統)及NFAT系統(參見例如Kallunki等人, Cells(2019) 8(8):796; Uchibori等人, Mol Ther Oncolytics.(2018) 12:16-25)。在一些實施例中，表現卡匣包括信號肽。在一些實施例中，信號肽包含SEQ ID NO: 2之胺基酸序列的編碼序列。 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.

在一些實施例中，表現卡匣亦包括Kozak序列、多腺苷酸化位點及促進編碼序列之轉錄及/或轉譯的其他元件。舉例而言，土拔鼠肝炎病毒轉錄後反應元件(WPRE)或其變異體可包括在表現卡匣之3'未轉譯區處。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.

在表現卡匣中，轉錄/轉譯調節元件，諸如啟動子、任何強化子及其類似物可操作地連接於編碼序列，以便允許編碼序列之有效表現及RNA轉錄物之有效轉譯。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.

在某些實施例中，本發明提供一種單一載體構築體(例如慢病毒載體)，其包含多順反子表現卡匣，該多順反子表現卡匣包含哺乳動物啟動子、c-Jun編碼序列、兩條抗GPC3 caTCR鏈之編碼序列、抗GPC3 CSR之編碼序列及聚腺苷酸化信號序列。編碼序列係藉由核苷酸連接子連接，該核苷酸連接子可為IRES或自裂解肽之編碼序列(例如P2A、T2A、E2A、F2A或其功能等效物)。藉助於實例， 圖 1A繪示此類表現卡匣，其中啟動子為EF-1α啟動子。 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.

在特定實施例中，表現卡匣編碼包含SEQ ID NO: 1之c-Jun、包含兩條分別包含SEQ ID NO: 30及31之多肽鏈的抗GPC3 caTCR及包含SEQ ID NO: 33之抗GPC3 CSR (例如 圖 1A中之構築體2)。構築體可為重組慢病毒載體且可進一步包含在EF-1α啟動子上游之中央聚嘌呤區(central polypurine tract，cPPT)及在CSR編碼序列與SV40聚腺苷酸化信號之間的WPRE序列或其他在哺乳動物細胞中有效轉導及表現之序列。 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.

表現卡匣中之編碼序列可經密碼子最佳化以用於所關注宿主細胞(例如人類細胞)中之最佳表現量。The coding sequence in the expression cassette can be codon-optimized for optimal expression in the host cell of interest (e.g., human cells).

編碼抗GPC3 caTCR、抗GPC3 CSR及c-Jun之核酸分子可整合至經工程改造之細胞的基因體中或保持游離。整合可為經由基因編輯發生的靶向整合(例如，由CRISPR、TALEN、鋅指核酸酶及巨核酸酶介導)。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).

經工程改造之細胞可藉由正向選擇技術富集。舉例而言，可根據其在例如流動式細胞測量術分析中結合GPC3的能力選擇細胞。為證實c-Jun表現，可對經工程改造之T細胞進行RT-PCT。正向選擇可引起細胞群中caTCR +CSR +c-Jun +細胞富集，其中三陽性T細胞構成總細胞群之超過30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%或95%。可低溫保存經工程改造之細胞直至使用。 E. 變異體 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

在一些實施例中，涵蓋本文所提供之抗GPC3 caTCR、抗GPC3 CSR及c-Jun的胺基酸序列變異體。在一些實施例中，涵蓋抗GPC3 caTCR之抗原結合模組及/或抗GPC3 CSR之配位體結合模組的胺基酸序列變異體。舉例而言，可能需要改善一或多種由表現構築體(例如抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun)編碼之多肽的結合親和力及/或其他生物特性。抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun之胺基酸序列變異體可藉由將適當修飾引入編碼抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun之核苷酸序列中或藉由肽合成來製備。此類修飾包括例如抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun之胺基酸序列內殘基之缺失及/或***及/或取代。可進行缺失、***及取代之任何組合以獲得最終構築體，其限制條件為最終構築體具有所需特徵，例如GPC3結合。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.

在一些實施例中，提供具有一或多個胺基酸取代之抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun變異體。用於取代型突變誘發之所關注位點包括高變區(HVR)及構架區(FR)。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).

保守取代展示於下 表 2中。 表 2. 保守取代 . 原始殘基 例示性取代 較佳取代 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；正白胺酸 Leu Leu (L) 正白胺酸；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；正白胺酸 Leu 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

胺基酸可根據共同的側鏈特性分成不同類別： a. 疏水性：正白胺酸、Met、Ala、Val、Leu、Ile； b. 中性親水性：Cys、Ser、Thr、Asn、Gln； c. 酸性：Asp、Glu； d. 鹼性：His、Lys、Arg； e. 影響鏈取向之殘基：Gly、Pro；及 f. 芳族：Trp、Tyr、Phe。 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.

非保守取代將引起此等類別中之一者的成員換成另一個類別。Non-conservative substitutions will result in exchanging a member of one of these classes for another class.

例示性取代變異體為由抗GPC3 caTCR之抗原結合模組或抗GPC CSR之配位體結合模組構成之親和力成熟抗體部分，其可例如使用基於噬菌體呈現之親和力成熟技術便利地產生。簡言之，使一或多個CDR殘基突變且在噬菌體上呈現變異抗體部分且針對特定生物活性(例如結合親和力)進行篩選。改變(例如取代)可發生於HVR中，以例如改善抗體部分親和力。此類改變可在HVR「熱點」(亦即由在體細胞成熟過程期間經歷高頻率突變之密碼子所編碼之殘基(參見例如Chowdhury, Methods Mol. Biol.207:179-196 (2008))，及/或特異性決定殘基(SDR))中進行，其中測試所得變異V H或V L之結合親和力。藉由構築二級庫及自二級庫再選擇來實現親和力成熟已描述於例如Hoogenboom等人, Methods in Molecular Biology178:1-37 (O'Brien等人編輯, Human Press, Totowa, NJ, (2001))中。 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)).

在親和力成熟之一些實施例中，藉由多種方法(例如易錯PCR、鏈改組或寡核苷酸定向突變誘發)中之任一者將多樣性引入為了成熟所選之可變基因中。隨後產生二級庫。接著篩選庫以鑑別具有所需親和力之任何抗體部分變異體。另一種引入多樣性之方法涉及HVR引導方法，其中將若干HVR殘基(例如，一次4-6個殘基)隨機分組。可特異性地鑑別抗原結合所涉及之HVR殘基，例如使用丙胺酸掃描突變誘發或模型化來鑑別。常常尤其以CDR-H3及CDR-L3為目標。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.

在一些實施例中，取代、***或缺失可發生在一或多個HVR內，只要此類改變不實質上減弱抗GPC3 caTCR及/或抗GPC3 CSR結合GPC3的能力即可。舉例而言，可在HVR中進行不實質上降低結合親和力之保守改變(例如如本文所提供之保守取代)。此類改變可在HVR「熱點」或SDR外。在上文所提供之變異VH及VL序列之某些實施例中，各HVR不變或含有不超過一個、兩個或三個胺基酸取代。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.

一種用於鑑別可靶向突變誘發之抗體(例如抗GPC3 caTCR之抗原結合模組或抗GPC3 CSR中所包含的抗體部分)之殘基或區的有用方法被稱作「丙胺酸掃描突變誘發」，如Cunningham及Wells (1989) Science, 244:1081-1085所述。在此方法中，鑑別殘基或一組目標殘基(例如帶電荷殘基，諸如arg、asp、his、lys及glu)且用中性或帶負電荷胺基酸(例如丙胺酸或聚丙胺酸)置換以確定抗體與抗原之相互作用是否受到影響。可在對初始取代展現功能敏感性之胺基酸位置處引入其他取代。替代地或另外，可測定抗原-抗體複合物之晶體結構以鑑別抗GPC3 caTCR及/或抗GPC3 CSR與GPC3之間的接觸點。此類接觸殘基及鄰近殘基可作為取代候選目標或排除在取代候選之外。可對變異體進行篩選以確定其是否展現所需特性。 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.

胺基酸序列***包括長度在一個殘基至含有一百個或更多個殘基之多肽範圍內的胺基端及/或羧基端融合，以及單個或多個胺基酸殘基之序列內***。末端***之實例包括具有N端甲硫胺醯基殘基之抗體部分(例如抗GPC3 caTCR之抗原結合模組及/或抗GPC3 CSR之配位體結合模組)。抗體部分之其他***變異體包括抗體部分之N端或C端與酶(例如用於抗體導向之酶前藥療法，ADEPT)或延長構築體(例如抗GPC3 caTCR及/或抗GPC3 CSR構築體)之血清半衰期的多肽的融合物。 糖基化變異體 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

在一些實施例中，改變本文所提供之抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun以增加或減少表現構築體糖基化之程度。表現構築體之糖基化位點的添加或缺失可藉由改變抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun構築體或其多肽部分之胺基酸序列以便產生或移除一或多個糖基化位點來便利地實現。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.

在抗GPC3 caTCR包含Fc區的情況下，可改變與其連接之碳水化合物。由哺乳動物細胞產生之天然抗體通常包含一般藉由N鍵連接於Fc區之CH2域之Asn297的分支鏈雙觸角寡醣(參見例如Wright等人, TIBTECH15:26-32 (1997))。寡醣可包括各種碳水化合物，例如甘露糖、N-乙醯基葡糖胺(GlcNAc)、半乳糖及唾液酸，以及連接於雙觸寡醣結構之「主幹」中之GlcNAc的海藻糖。在一些實施例中，可以修飾本發明之抗GPC3構築體中的寡醣以便產生某些特性改良的抗GPC3構築體變異體。 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.

連接於Fc之CH2域的N-聚糖為異質的。CHO細胞中產生的抗體或Fc融合蛋白藉由海藻糖基轉移酶活性進行海藻糖基化。參見Shoji-Hosaka等人, J. Biochem. 2006, 140:777- 83。通常，可在人類血清中偵測到小百分比之天然存在之去海藻糖基化IgG。Fc之N-糖基化對於結合FcγR至關重要；且N-聚糖之去海藻糖基化提高FcγRIIIa之Fc的結合能力。增強的FcγRIIIa結合可以增強ADCC，此在需要細胞毒性的某些抗體治療應用中可為有利的。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.

在一些實施例中，當Fc介導之細胞毒性非所需時，增強的效應功能可為有害的。在一些實施例中，Fc片段或CH2域未經糖基化。在一些實施例中，CH2域中之N-糖基化位點發生突變，阻止了糖基化。 經半胱胺酸工程改造之變異體 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

在一些實施例中，可能需要產生經半胱胺酸工程改造之抗GPC3 caTCR構築體、抗GPC3 CSR構築體及/或c-Jun多肽，其中一或多個胺基酸殘基經半胱胺酸殘基取代。在一些實施例中，經取代之殘基存在於本文所述之構築體之可接入位點處。藉由用半胱胺酸取代彼等殘基，從而使反應性硫醇基位於構築體之可接入位點且可用於使抗GPC3構築體與其他部分結合。經半胱胺酸工程改造之表現構築體可如例如美國專利第7,521,541號中所述產生。 III. 免疫細胞 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

在一個態樣中，本發明提供表現抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun多肽(諸如本文所述之抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun多肽中之任一者)的經修飾細胞(諸如免疫細胞，例如T細胞)。本文提供製備表現抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun多肽(諸如本文所述之抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun多肽中之任一者)的經修飾細胞(諸如T細胞)的例示性方法。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.

在一些實施例中，本發明提供一種免疫細胞(諸如T細胞)，其在其表面上呈遞根據本文所描述之caTCR、CSR及c-Jun多肽中之任一者的caTCR、CSR及c-Jun多肽(此類免疫細胞在本文中亦被稱為「caTCR加CSR加c-Jun免疫細胞」)。本發明之caTCR及CSR特異性靶向GPC3 (此類caTCR及CSR在本文中亦分別稱為「抗GPC3 caTCR」及「抗GPC3 CSR」)。在一些實施例中，免疫細胞包含編碼抗GPC3 caTCR及抗GPC3 CSR的核酸，其中抗GPC3 caTCR及抗GPC3 CSR由核酸表現且定位於免疫細胞表面。在一些實施例中，免疫細胞為T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，免疫細胞不表現抗GPC3 caTCR之TCR-TM所源自之TCR次單元。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入抗GPC3 caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或T細胞為γδ T細胞且所引入抗GPC3 caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少免疫細胞之內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。用於破壞基因表現之細胞修飾包括此項技術中已知的任何此類技術，包括例如RNA干擾(例如，siRNA、shRNA、miRNA)、基因編輯(例如，基於CRISPR或TALEN之基因剔除)及其類似技術。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.

舉例而言，在一些實施例中，提供一種免疫細胞(諸如T細胞)，其包含編碼根據本文所述之抗GPC3 caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的人類c-Jun多肽的核酸，其中抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun多肽由核酸表現且定位於免疫細胞表面。在一些實施例中，核酸包含編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列、編碼抗GPC3 caTCR之第二caTCR多肽鏈的第二caTCR核酸序列、編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列及編碼人類c-Jun多肽鏈的人類c-Jun核酸序列。在一些實施例中，第一抗GPC3 caTCR核酸序列及第二抗GPC3 CSR核酸序列及人類c-Jun核酸序列各自包含於不同載體中。在一些實施例中，一些或全部核酸序列包含於同一載體中。載體可選自例如由哺乳動物表現載體及病毒載體(諸如源於反轉錄病毒、腺病毒、腺相關病毒、疱疹病毒及慢病毒之彼等載體)組成之群。在一些實施例中，將一或多種載體整合於免疫細胞之宿主基因體中。在一些實施例中，第一抗GPC3 caTCR核酸序列及第二抗GPC3 CSR核酸序列及人類c-Jun核酸序列各自處於不同啟動子的控制下。在一些實施例中，啟動子中的一些或全部具有相同序列。在一些實施例中，一些或全部啟動子具有不同序列。在一些實施例中，一些或全部核酸序列處於單一啟動子之控制下。在一些實施例中，一些或全部啟動子為誘導性的。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。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.

因此，在一些實施例中，提供一種抗GPC3 caTCR加抗GPC3 CSR加c-Jun (「caTCR+CSR+c-Jun」)免疫細胞(諸如T細胞)，其在其表面上表現根據本文所述之抗GPC3 caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的人類c-Jun多肽，其中caTCR+CSR+c-Jun免疫細胞包含a)編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列；b)編碼抗GPC3 caTCR之第二抗GPC3 caTCR多肽鏈的第二抗GPC3 caTCR核酸序列；c)編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列；及d)編碼c-Jun多肽的c-Jun核酸序列，其中第一及第二抗GPC3 caTCR多肽鏈由第一及第二抗GPC3 caTCR核酸序列表現，形成抗GPC3 caTCR，其中抗GPC3 CSR多肽鏈由抗GPC3 CSR核酸表現，形成抗GPC3 CSR，其中c-Jun多肽由c-Jun核酸表現，形成c-Jun多肽，且其中抗GPC3 caTCR與抗GPC3 CSR定位於免疫細胞表面。在一些實施例中，第一抗GPC3 caTCR核酸序列包含於第一載體(諸如慢病毒載體)中，第二抗GPC3 caTCR核酸序列包含於第二載體(諸如慢病毒載體)中，抗GPC3 CSR核酸序列包含於第三載體(諸如慢病毒載體)中，且c-Jun核酸序列包含於第四載體(諸如慢病毒載體)中。在一些實施例中，第一抗GPC3 caTCR核酸序列及第二抗GPC3 CSR核酸序列中的一些或全部包含於同一載體(諸如慢病毒載體)中。在一些實施例中，第一及第二抗GPC3 caTCR核酸序列、抗GPC3 CSR核酸序列及c-Jun核酸序列中的一些或全部包含於同一載體(諸如慢病毒載體)中。在一些實施例中，第一及第二抗GPC3 caTCR核酸序列、抗GPC3 CSR核酸序列及c-Jun核酸序列各自可操作地連接於啟動子。在一些實施例中，一些或全部核酸序列處於單一啟動子之控制下。在一些實施例中，啟動子中的一些或全部具有相同序列。在一些實施例中，一些或全部啟動子具有不同序列。在一些實施例中，一些或全部啟動子為誘導性的。在一些實施例中，一些或全部載體為病毒載體(諸如慢病毒載體)。在一些實施例中，免疫細胞不表現衍生caTCR之TCR-TM之TCR子單位。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或免疫細胞為γδ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少其內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，一些或全部載體為整合於免疫細胞之宿主基因體中之病毒載體(諸如慢病毒載體)。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.

在一些實施例中，提供一種caTCR+CSR+c-Jun免疫細胞(諸如T細胞)，其在其表面上表現根據本文所述之caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的人類c-Jun多肽，其中caTCR+CSR+c-Jun免疫細胞包含a)第一載體，其包含第一啟動子，該第一啟動子可操作地連接於編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列；b)第二載體，其包含第二啟動子，該第二啟動子可操作地連接於編碼抗GPC3 caTCR之第二抗GPC3 caTCR多肽鏈的第二抗GPC3 caTCR核酸序列；c)第三載體，其包含第三啟動子，該第三啟動子可操作地連接於編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列；及d)第四載體，其包含第四啟動子，該第四啟動子可操作地連接於編碼c-Jun多肽之c-Jun核酸序列，其中第一及第二抗GPC3 caTCR多肽鏈由第一及第二抗GPC3 caTCR核酸序列表現，形成抗GPC3 caTCR，抗GPC3 CSR多肽鏈由抗GPC3 CSR核酸序列表現，形成抗GPC3 CSR，c-Jun多肽由c-Jun核酸序列表現，形成c-Jun多肽，且其中caTCR及CSR定位於免疫細胞表面。在一些實施例中，啟動子中的一些或全部具有相同序列。在一些實施例中，一些或全部啟動子具有不同序列。在一些實施例中，一些或全部啟動子為誘導性的。在一些實施例中，免疫細胞不表現衍生caTCR之TCR-TM之TCR子單位。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或免疫細胞為γδ T細胞且所引入caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少其內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，第一及第二載體為整合於免疫細胞之宿主基因體中之病毒載體(諸如慢病毒載體)。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.

在一些實施例中，提供一種caTCR+CSR+c-Jun免疫細胞(諸如T細胞)，其在其表面上表現根據本文所述之caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的c-Jun多肽，其中caTCR+CSR+c-Jun免疫細胞包含a)第一載體，其包含i)第一啟動子，該第一啟動子可操作地連接於編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列；及ii)第二啟動子，該第二啟動子可操作地連接於編碼抗GPC3 caTCR之第二抗GPC3 caTCR多肽鏈的第二抗GPC3 caTCR核酸序列；b)第二載體，其包含第三啟動子，該第三啟動子可操作地連接於編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列；及c)第三載體，其包含第四啟動子，該第四啟動子可操作地連接於編碼c-Jun多肽之c-Jun核酸序列，其中第一及第二抗GPC3 caTCR多肽鏈由第一及第二抗GPC3 caTCR核酸序列表現，形成抗GPC3 caTCR，抗GPC3 CSR多肽鏈由抗GPC3 CSR核酸序列表現，形成抗GPC3 CSR，c-Jun多肽由c-Jun核酸序列表現，且其中抗GPC3 caTCR定位於免疫細胞表面。在一些實施例中，啟動子中的一些或全部具有相同序列。在一些實施例中，一些或全部啟動子具有不同序列。在一些實施例中，一些或全部啟動子為誘導性的。在一些實施例中，免疫細胞不表現抗GPC3 caTCR之TCR-TM所源自之TCR次單元。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入之caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或免疫細胞為γδ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少其內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，第一及第二載體為整合於免疫細胞之宿主基因體中之病毒載體(諸如慢病毒載體)。應瞭解，亦涵蓋核酸序列中之任一者經交換的實施例，諸如其中第一或第二抗GPC3 caTCR核酸序列經抗GPC3 CSR核酸序列或c-Jun核酸序列交換。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.

在一些實施例中，提供一種caTCR+CSR+c-Jun免疫細胞(諸如T細胞)，其在其表面上表現根據本文所述之caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的c-Jun多肽，其中caTCR+CSR+c-Jun免疫細胞包含a)第一載體，其包含i)編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列，及ii)編碼抗GPC3 caTCR之第二抗GPC3 caTCR多肽鏈的第二抗GPC3 caTCR核酸序列，其中第一及第二抗GPC3 caTCR核酸序列處於第一啟動子的控制下；b)第二載體，其包含第二啟動子，該第二啟動子可操作地連接於編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列；及c)第三載體，其包含第三啟動子，該第三啟動子可操作地連接於編碼c-Jun多肽之c-Jun核酸序列，其中第一及第二抗GPC3 caTCR多肽鏈由第一及第二抗GPC3 caTCR核酸序列表現，形成抗GPC3 caTCR，抗GPC3 CSR多肽鏈由抗GPC3 CSR核酸序列表現，形成抗GPC3 CSR，c-Jun多肽由c-Jun核酸序列表現，且其中抗GPC3 caTCR及抗GPC3 CSR定位於免疫細胞表面。在一些實施例中，第一啟動子可操作地連接於第一抗GPC3 caTCR核酸序列之5'端，且存在選自由內部核糖體進入位點(IRES)及編碼自裂解2A肽(諸如P2A、T2A、E2A或F2A)之核酸組成之群的核酸連接子將第一抗GPC3 caTCR核酸序列之3'端與第二抗GPC3 caTCR核酸序列之5'端連接，其中第一抗GPC3 caTCR核酸序列及第二抗GPC3 caTCR核酸序列在啟動子的控制下轉錄成單一RNA。在一些實施例中，第一啟動子可操作地連接於第二抗GPC3 caTCR核酸序列之5'端，且存在選自由內部核糖體進入位點(IRES)及編碼自裂解2A肽(諸如P2A、T2A、E2A或F2A)之核酸組成之群的核酸連接子將第二抗GPC3 caTCR核酸序列之3'端與第一抗GPC3 caTCR核酸序列之5'端連接，其中第一抗GPC3 caTCR核酸序列及第二抗GPC3 caTCR核酸序列在啟動子的控制下轉錄成單一RNA。在一些實施例中，第一及/或第二啟動子具有相同序列。在一些實施例中，第一及/或第二啟動子具有不同序列。在一些實施例中，第一及/或第二啟動子為可誘導的。在一些實施例中，免疫細胞不表現衍生caTCR之TCR-TM之TCR子單位。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或免疫細胞為γδ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少其內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，載體為整合於免疫細胞之宿主基因體中之病毒載體(諸如慢病毒載體)。應瞭解，亦涵蓋核酸序列中之任一者經交換的實施例，諸如其中第一或第二抗GPC3 caTCR核酸序列經抗GPC3 CSR核酸序列或c-Jun核酸序列交換。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.

在一些實施例中，提供一種caTCR+CSR+c-Jun免疫細胞(諸如T細胞)，其在其表面上表現根據本文所述之caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及根據本文所述之c-Jun多肽中之任一者的c-Jun多肽，其中caTCR+CSR+c-Jun免疫細胞包含一種載體，其包含：a)編碼抗GPC3 caTCR之第一抗GPC3 caTCR多肽鏈的第一抗GPC3 caTCR核酸序列；b)編碼抗GPC3 caTCR之第二抗GPC3 caTCR多肽鏈的第二抗GPC3 caTCR核酸序列；c)編碼抗GPC3 CSR之抗GPC3 CSR多肽鏈的抗GPC3 CSR核酸序列；及d)編碼c-Jun多肽之c-Jun核酸序列，其中第一及第二抗GPC3 caTCR核酸序列、抗GPC3 CSR核酸序列及c-Jun多肽處於單一啟動子的控制下；其中第一及第二抗GPC3 caTCR多肽鏈由第一及第二抗GPC3 caTCR核酸序列表現，形成抗GPC3 caTCR，抗GPC3 CSR多肽鏈由抗GPC3 CSR核酸序列表現，形成抗GPC3 CSR，c-Jun多肽由c-Jun核酸序列表現，且其中抗GPC3 caTCR及抗GPC3 CSR定位於免疫細胞表面。在一些實施例中，啟動子可操作地連接於一個核酸序列，該核酸序列藉由個別地選自由內部核糖體進入位點(IRES)及編碼自裂解2A肽(諸如P2A、T2A、E2A或F2A)之核酸組成之群的核酸連接子連接於其他核酸序列，使得第一及第二caTCR核酸序列以及CSR核酸序列在啟動子之控制下轉錄為單一RNA。在一些實施例中，啟動子為可誘導的。在一些實施例中，免疫細胞不表現抗GPC3 caTCR之TCR-TM所源自之TCR次單元。舉例而言，在一些實施例中，免疫細胞為αβ T細胞且所引入之抗GPC3 caTCR之TCR-TM包含源於TCR δ及γ鏈之序列，或免疫細胞為γδ T細胞且所引入之caTCR之TCR-TM包含源於TCR α及β鏈之序列。在一些實施例中，免疫細胞經修飾以阻斷或減少其內源性TCR子單位中之一者或兩者之表現。舉例而言，在一些實施例中，免疫細胞為經修飾以阻斷或減少TCR α及/或β鏈之表現的αβ T細胞，或免疫細胞為經修飾以阻斷或減少TCR γ及/或δ鏈之表現的γδ T細胞。在一些實施例中，免疫細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。在一些實施例中，載體為整合於免疫細胞之宿主基因體中之病毒載體(諸如慢病毒載體)。 A. 免疫細胞來源 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

本發明之經工程改造之免疫細胞的來源可以為待治療之患者(即自體細胞)或來自並非待治療患者之供體(例如同種異體細胞)。在一些實施例中，經工程改造之免疫細胞為經工程改造之T細胞。本文中之經工程改造之T細胞可為CD4 +CD8 -(亦即CD4單陽性) T細胞、CD4 -CD8 +(亦即CD8單陽性) T細胞或CD4 +CD8 +(雙陽性) T細胞。功能上，T細胞可為細胞毒性T細胞、輔助性T細胞、自然殺手T細胞、抑制性T細胞或其混合物。待工程改造之T細胞可為自體或同種異體。 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.

在T細胞擴增及基因修飾之前，T細胞來源獲自個體。T細胞可獲自許多來源，包括周邊血液單核細胞、骨髓、淋巴結組織、臍帶血、胸腺組織、來自感染位點之組織、腹水、肋膜積液、脾組織及腫瘤。在本發明之一些實施例中，可以使用此項技術中可利用的任何數目個T細胞株。在本發明之一些實施例中，T細胞可獲自使用熟習此項技術者已知之任意數目之技術(諸如Ficoll™分離)自個體收集之血液單位。在一些實施例中，藉由血球分離術獲得來自個體之循環血液的細胞。血球分離術產物通常含有淋巴細胞，包括T細胞、單核球、粒細胞、B細胞、其他成核白血球、紅血球及血小板。在一些實施例中，藉由血球分離術收集之細胞可經洗滌以移除血漿部分且將細胞置於適當緩衝液或培養基中以用於後續處理步驟。在一些實施例中，細胞用磷酸鹽緩衝鹽水(PBS)進行洗滌。在一些實施例中，洗滌溶液缺乏鈣且可能缺乏鎂或可能缺乏許多(若非全部)二價陽離子。如一般熟習此項技術者將容易瞭解，洗滌步驟可藉由熟習此項技術者已知的方法實現，諸如藉由根據製造商說明書使用半自動「流通」離心(例如Cobe 2991細胞處理器、Baxter CytoMate或Haemonetics細胞保存器5)。洗滌之後，可以將細胞再懸浮於多種生物相容性緩衝液中，諸如無Ca 2+、無Mg 2+之PBS、PlasmaLyte A或其他具有或不具有緩衝劑之鹽水溶液。替代地，可移除血球分離術樣品之非所要組分且將細胞直接再懸浮於培養基中。 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.

在一些實施例中，藉由溶解紅血球及耗乏單核球而使T細胞與周邊血液淋巴細胞分離，例如藉由PERCOLL™梯度進行離心或藉由逆流離心淘析。T細胞之特定亞群(諸如CD3 +、CD28 +、CD4 +、CD8 +、CD45RA +及CD45RO +T細胞)可藉由正向或負向選擇技術進一步分離。舉例而言，在一些實施例中，T細胞藉由持續對於所需T細胞之正向選擇而言足夠的時間段與抗CD3/抗CD28 (亦即3×28)結合珠粒，諸如DYNABEADS ®M-450 CD3/CD28 T一起培育而分離。在一些實施例中，該時間段為約30分鐘。在一些實施例中，該時間段的範圍為30分鐘至36小時或更長及其間的所有整數值。在一些實施例中，該時間段為至少1、2、3、4、5或6小時。在一些實施例中，該時間段為10至24小時。在一些實施例中，培育時間段為24小時。為了自白血病患者分離出T細胞，使用較長培育時間(諸如24小時)可提高細胞產量。在T細胞相較於其他細胞類型而言很少的任何情形下，可以使用較長培育時間分離T細胞，諸如自腫瘤組織或自免疫功能不全個體分離出腫瘤浸潤淋巴細胞(TIL)。另外，使用較長培育時間可提高捕捉CD8 +T細胞之效率。因此，藉由僅縮短或延長允許T細胞結合CD3/CD28珠粒之時間及/或藉由增加或減少珠粒與T細胞的比率，可以在培養起始或在製程期間之其他時間點藉此優先選擇T細胞亞群。另外，藉由增加或降低珠粒或其他表面上抗CD3及/或抗CD28抗體之比率，對於或針對在培養起始時或在其他所要時間點可優先選擇T細胞亞群。熟習此項技術者將認識到在本發明之背景下亦可使用多輪選擇。在一些實施例中，在活化及擴增過程中可能需要執行選擇程序且使用「未選擇之」細胞。「未選擇之」細胞亦可經受其他多輪選擇。 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.

藉由負向選擇富集T細胞群可使用針對負向選擇之細胞所獨特之表面標記物的抗體之組合來實現。一種方法為經由負磁性免疫黏附或流動式細胞測量術分選及/或選擇細胞，該負磁性免疫黏附或流動式細胞測量術使用針對負向選擇細胞上存在之細胞表面標記物之單株抗體混合物。舉例而言，為了藉由負向選擇富集CD4+細胞，單株抗體混合液通常包括針對CD-14、CD20、CD11b、CD-16、HLA-DR及CD8之抗體。在一些實施例中，可能需要富集或正向選擇通常表現CD4 +、CD25 +、CD62Lhi、GITR +及FoxP3 +之調節性T細胞。替代地，在一些實施例中，T調節細胞藉由抗CD25結合之珠粒或其他類似選擇方法耗乏。 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.

為了藉由正向選擇或負向選擇分離所需細胞群，細胞濃度及表面(例如粒子，諸如珠粒)可變化。在一些實施例中，可能需要顯著減小珠粒與細胞混合在一起之體積(亦即增加細胞濃度)，以確保細胞與珠粒之最大接觸。舉例而言，在一些實施例中，使用約20億個細胞/毫升之濃度。在一些實施例中，使用約10億個細胞/毫升之濃度。在一些實施例中，使用大於約1億個細胞/毫升。在一些實施例中，使用約1000萬、1500萬、2000萬、2500萬、3000萬、3500萬、4000萬、4500萬或5000萬個細胞/毫升中之任一者之細胞濃度。在一些實施例中，使用約7500萬、8000萬、8500萬、9000萬、9500萬或1億個細胞/毫升中之任一者的細胞濃度。在一些實施例中，使用約1.25億或約1.50億個細胞/毫升之濃度。使用高濃度可以引起細胞產量、細胞活化及細胞擴增增加。另外，使用高細胞濃度可以更有效地捕捉可能微弱表現所關注目標抗原的細胞，諸如CD28陰性T細胞，或來自存在許多腫瘤細胞之樣品(亦即，白血病血液、腫瘤組織等)的細胞。此類細胞群可具有治療價值且需要獲得。舉例而言，使用高細胞濃度允許更有效選擇通常具有較弱CD28表現之CD8 +T細胞。 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.

在本發明之一些實施例中，T細胞直接獲自治療之後的患者。就此而言，已觀測到在某些癌症治療之後，尤其使用破壞免疫系統之藥物治療之後，在患者通常將自治療恢復的時段期間治療之後不久，獲得之T細胞品質可最佳或其離體擴增之能力得到改良。同樣，在使用本文所述之方法離體操縱之後，此等細胞可處於較佳狀態，從而增強移植及活體內擴增。因此，本發明之情形內涵蓋在此恢復階段期間收集血球，包括T細胞、樹突狀細胞或造血譜系之其他細胞。此外，在一些實施例中，移動(例如隨GM-CSF移動)及調理療法可以用於建立個體內之條件，其中有利於重建、再循環、再生及/或擴增特定細胞類型，尤其在治療之後的限定時間窗期間。說明性細胞類型包括T細胞、B細胞、樹突狀細胞及免疫系統之其他細胞。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.

不管是在對T細胞進行基因修飾以表現所需表現構築體之前還是在此之後，一般均可使用如例如美國專利第6,352,694號；第6,534,055號；第6,905,680號；第6,692,964號；第5,858,358號；第6,887,466號；第6,905,681號；第7,144,575號；第7,067,318號；第7,172,869號；第7,232,566號；第7,175,843號；第5,883,223號；第6,905,874號；第6,797,514號；第6,867,041號；及美國專利申請公開案第20060121005號中所描述之方法活化及擴增T細胞。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.

一般而言，本發明之T細胞係藉由使刺激CD3/TCR複合物相關信號的藥劑及刺激T細胞表面上之協同刺激分子的配位體與其所連接之表面接觸來擴增。具體言之，T細胞群可經如下刺激：諸如藉由與抗CD3抗體或其抗原結合片段或固定於表面上之抗CD3抗體接觸，或藉由與結合鈣離子載體之蛋白激酶C活化因子(例如苔蘚蟲素)接觸。為了協同刺激T細胞表面上之輔助分子，使用結合輔助分子之配位體。舉例而言，可使T細胞群與抗CD3抗體及抗CD28抗體在適於刺激T細胞增殖之條件下接觸。為了刺激CD4 +T細胞或CD8 +T細胞之增殖，可使用抗CD3抗體及抗CD28抗體。抗CD28抗體之實例包括9.3、B-T3、XR-CD28 (Diaclone, Besançon, France)，可按此項技術中通常已知之其他方法使用(Berg等人, Transplant Proc. 30(8):3975-3977, 1998; Haanen等人, J. Exp. Med. 190(9):13191328, 1999; Garland等人, J. Immunol. Meth. 227(1-2):53-63, 1999)。 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).

細胞培養條件可以包括以下中的一或多種：特定培養基、溫度、氧含量、二氧化碳含量、時間、試劑，例如營養素、胺基酸、抗生素、離子及/或刺激因子，諸如細胞介素、趨化因子、抗原、結合搭配物、融合蛋白、重組可溶性受體及任何其他旨在激活細胞之試劑。在一些實施例中，培養條件包括添加IL-2、IL-7及/或IL-15。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.

在一些實施例中，待工程改造之細胞可為在工程改造之後分化為成熟T細胞之多能或多潛能細胞。此等非T細胞可為同種異體的，且可為例如人類胚胎幹細胞、人類誘導多能幹細胞或造血幹細胞或先驅細胞。為了易於描述，多能及多潛能細胞在本文中共同稱作「先驅細胞」。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."

在使用同種異體細胞之情況下，較佳對同種異體細胞進行工程改造以減少移植物抗宿主排斥反應(例如藉由基因剔除內源性 B2M及/或 TRAC基因)。 B. 免疫細胞或祖細胞之工程改造 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

在一些態樣中，免疫細胞之細胞工程改造包含病毒基因工程改造、非病毒基因工程改造、引入受體以允許腫瘤特異性靶向(例如抗GPC3 caTCR及抗GPC3 CSR)、引入改良T細胞功能之一或多種內源性基因、引入改良免疫細胞(例如T細胞)功能的一或多種合成基因(例如編碼c-Jun多肽的聚核苷酸，使得免疫細胞與尚未修飾之對應細胞相比展現增加的c-Jun表現)或其任何組合。如本發明中的其他地方進一步描述的，在一些態樣中，可以用轉錄活化因子(例如，基於CRISPR/Cas系統之轉錄活化因子)對細胞進行工程改造或修飾，其中該轉錄活化因子能夠誘導及/或增加所關注蛋白質(例如，c-Jun)的內源性表現。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).

在一些態樣中，本文所描述之細胞已經轉錄活化因子修飾，該轉錄活化因子能夠誘導及/或增加所關注蛋白質(例如，c-Jun)在細胞中之內源性表現。如本文所用，術語「轉錄活化因子」係指增加基因或基因體之轉錄(例如藉由結合核酸序列之強化子或啟動子近端元件，藉此誘導其轉錄)的蛋白質。可與本發明一起使用之此類轉錄活化因子的非限制性實例包括：基於轉錄活化因子樣效應子(TALE)之轉錄活化因子、基於鋅指蛋白(ZFP)之轉錄活化因子、基於成簇規律間隔短回文重複序列(CRISPR)/CRISPR相關蛋白(Cas)系統之轉錄活化因子或其組合。參見例如Kabadi等人, Methods(2014) 69(2):188-197，其以全文引用的方式併入本文中。 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.

在一些態樣中，本文所述之細胞已經用基於CRISPR/Cas系統之轉錄活化因子，諸如CRISPR活化(CRISPRa)修飾。參見例如Nissim等人, Molecular Cell(2014) 54(4):698-710；Perez-Pinera等人, Nat. Methods(2013) 10(10):973-976；Maeder等人, Nat. Methods(2013) 10(10):977-979；Cheng等人, Cell Res. (2013) 23(10):1163-71；Farzadfard等人, ACS Synth. Biol.(2013) 2(10):604-13；其全部皆以全文引用的方式併入本文中。CRISPRa為一種類型之CRISPR工具，其包含使用缺乏核酸內切酶活性但保持結合其引導RNA及目標DNA核酸序列之能力的經修飾Cas蛋白。可用於本發明之此類經修飾Cas蛋白質之非限制性實例為此項技術中已知的(參見例如Pandelakis等人, Cell Systems(2020) 10(1):1-14，其以全文引用之方式併入本文中)。在一些態樣中，經修飾Cas蛋白包含經修飾Cas9蛋白(在此項技術中亦稱為「dCas9」)。在一些態樣中，經修飾Cas蛋白包含經修飾Cas12a蛋白質。在一些態樣中，適用於本發明之經修飾Cas蛋白結合引導聚核苷酸(例如小引導RNA) (「經修飾Cas引導複合物」)，其中該引導聚核苷酸包含與編碼所關注蛋白質(例如c-Jun)之核酸序列之區域互補的識別序列。在某些態樣中，引導聚核苷酸包含與編碼所關注蛋白質之內源性核酸序列的啟動子區互補的識別序列。在一些態樣中，一或多種轉錄活化因子附接至經修飾Cas引導複合物(例如經修飾Cas蛋白之N端及/或C端)，使得當經修飾Cas引導複合物引入細胞中時，一或多種轉錄活化因子可結合內源性基因之調節元件(例如啟動子區)，藉此誘導及/或增加編碼蛋白質(例如c-Jun)的表現。可使用之常見一般活化因子之說明性實例包括RNAP、VP16、VP64及p65之ω次單元(參見例如Kabadi及Gersbach, Methods(2014)69(2)：188-97)。 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).

在一些態樣中，一或多種轉錄抑制因子(例如Kruppel相關框域(KRAB))可附接至經修飾Cas引導複合物(例如經修飾Cas蛋白之N端及/或C端)，使得當引入細胞中時，該一或多種轉錄抑制因子可抑制或減少基因之轉錄，例如可干擾c-Jun表現之彼等基因(例如Bach2)。參見例如US20200030379A1及Yang等人, J Transl Med. (2021) 19:459，其各自以全文引用的方式併入本文中。在一些態樣中，適用於本發明之經修飾Cas蛋白可附接至一或多種轉錄活化因子與一或多種轉錄抑制因子兩者。 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.

不受任一理論束縛，在一些態樣中，使用此類經修飾Cas蛋白可允許所關注基因之條件性轉錄及表現。舉例而言，在一些態樣中，細胞(例如T細胞)經修飾以包含與蛋白酶(例如菸草蝕刻病毒(tobacco etch virus，TEV))連接之重組抗原受體(例如抗GPC3 caTCR及抗GPC3 CSR)及靶向c-Jun之啟動子區的單一引導RNA (sgRNA)。在一些態樣中，細胞經修飾以進一步包含用於活化T細胞之連接子(LAT)，該連接子與經修飾Cas蛋白複合，該經修飾Cas蛋白經由連接子(例如，TEV可裂解連接子)附接至轉錄活化因子(例如dCas9-VP64-p65-Rta轉錄活化因子(VPR))。在抗原受體活化後，經修飾Cas蛋白經釋放用於核定位且條件性地且可逆地誘導c-Jun之表現。參見例如Yang等人, J Immunother Cancer(2021) 9(增刊2):A164，其以全文引用的方式併入本文中。 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.

如對於熟習此項技術者而言將顯而易見的是，在一些態樣中，本文所描述之細胞已使用諸多方法之組合進行修飾。舉例而言，在一些態樣中，細胞已經修飾以包含(i)編碼一或多種蛋白質(例如抗GPC3 caTCR、抗GPC3 CSR及截短EGFR (EGFRt))的外源性核苷酸序列及(ii)增加內源性蛋白質(例如c-Jun)表現的外源性轉錄活化因子(例如CRISPRa)。在一些態樣中，細胞經修飾過以包含(i)編碼第一蛋白(例如抗GPC3 caTCR)的外源性核苷酸序列、(ii)編碼第二蛋白質(例如抗GPC3 CSR)的外源性核苷酸序列及(iii)編碼蛋白質(例如c-Jun蛋白)的外源性核苷酸序列。在一些態樣中，經修飾細胞可進一步包含編碼第三蛋白(例如，EGFRt)之外源性核苷酸序列。如本文所述，在一些態樣中，編碼第一、第二及第三蛋白之外源性核苷酸序列可為單一多順反子載體的一部分。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.

除非另外指示，否則可使用此項技術中已知之任何適合方法將一或多種外源性核苷酸序列及/或轉錄活化因子引入細胞中。用於向細胞遞送一或多種外源性核苷酸序列之適合方法的非限制性實例包括：轉染(亦稱為轉型及轉導)、電穿孔、非病毒遞送、病毒轉導、脂質奈米粒子遞送及其組合。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.

在一些態樣中，細胞已經用轉錄活化因子(例如基於CRISPR/Cas系統之轉錄活化因子，例如CRISPRa)修飾，使得內源性c-Jun蛋白之表現與尚未用轉錄活化因子修飾之對應細胞相比增加。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.

雖然本文提供的某些揭示內容大體上關於修飾免疫細胞以包含編碼c-Jun蛋白(野生型c-Jun或其變異體)的外源性核苷酸序列，但對於本領域技術人員而言將顯而易見的是，可使用其他適合的方法在細胞中誘導及/或增加c-Jun蛋白表現(野生型或其變異體)。舉例而言，如本文所描述，在一些態樣中，可用轉錄活化因子(例如，CRISPRa)增加內源性c-Jun蛋白表現。除非另外指示，否則使用外源性核苷酸序列之本文所提供之揭示內容同樣適用於本文所提供之在細胞中誘導及/或增加c-Jun蛋白表現的其他方法(例如轉錄活化因子，例如CRISPRa)。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).

本文中之免疫細胞(例如，T細胞)或祖細胞可經工程改造以表現抗GPC3 caTCR及抗GPC3 CSR，且過度表現c-Jun (例如，人類c-Jun多肽)。抗GPC3 caTCR可以特異性結合GPC3腫瘤細胞，且抗GPC3 CSR可特異性結合腫瘤細胞上的GPC3 (例如同一GPC3，但不同GPC3抗原決定基)。如本文所用，當結合之K D為≤100 nM，例如≤10 nM或≤1 nM時，受體(例如抗GPC3 caTCR或抗GPC3 CSR)被稱為特異性結合GPC3。K D結合親和力常數可例如藉由表面電漿子共振(使用例如Biacore™或Octet™系統)量測。 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).

在一些實施例中，免疫細胞表現具有均勻細胞表面分佈之抗GPC3 caTCR。在一些實施例中，免疫細胞表現具有均勻細胞表面分佈之抗GPC3 CSR。均勻細胞表面分佈可例如藉由具有連續外觀及均勻厚度或信號強度之染色圖案表徵。舉例而言，在一些實施例中，包含抗GPC3 caTCR、抗GPC3 CSR及人類c-Jun免疫細胞的組合物(諸如醫藥組合物)包含少於約10% (諸如少於約10%、9%、8%、7%、6%、5%、4%、3%、2%或1%)的細胞有抗GPC3 caTCR及/或抗GPC3 CSR在細胞表面上聚集。聚集可藉由例如具有不均勻厚度或信號強度的染色圖案或不連續、塊狀、點狀及/或不均勻分佈圖案表徵。在一些實施例中，caTCR+CSR+c-Jun T細胞表現抗GPC3 caTCR及/或抗GPC3 CSR，其中抗GPC3 caTCR及/或抗GPC3 CSR在細胞表面上的聚集量少於約10% (諸如小於約10%、9%、8%、7%、6%、5%、4%、3%、2%或1%)。在一些實施例中，抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞表現抗GPC3 caTCR及/或抗GPC3 CSR，其中抗GPC3 caTCR及/或抗GPC3 CSR在細胞表面上的聚集量少於約10% (諸如小於約10%、9%、8%、7%、6%、5%、4%、3%、2%或1%)。在一些實施例中，抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞具有低水平的抗原非依賴性抗GPC3 caTCR及/或抗GPC3 CSR活化。在一些實施例中，抗GPC3 caTCR T細胞具有低水平的抗原非依賴性抗GPC3 caTCR及/或抗GPC3 CSR活化。在一些實施例中，抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞具有低水平的T細胞耗竭。T細胞耗竭在延長之免疫活化條件期間自然發生，諸如在癌症或慢性感染情況下，其中T細胞變得功能異常。T細胞耗竭的特徵可為相比於功能效應子或記憶T細胞，效應功能減弱、抑制受體之表現延長及/或轉錄狀態改變。T細胞耗竭阻礙了腫瘤細胞及感染之最佳清除。抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞的T細胞耗竭可藉由此項技術中已知的任何方式表徵，例如藉由測定其功能及/或表型概況(Wherry, E. J., Nature immunology12(6): 492-499, 2011; Jiang, Y.等人, Cell death & disease6(6): e1792, 2015)。舉例而言，在一些實施例中，抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞表現低含量之一或多種T細胞耗竭標記物，包括例如PD-1、LAG-3、TIM-3、CTLA-4及BTLA。在一些實施例中，抗GPC3 caTCR加抗GPC3 CSR加人類c-Jun T細胞維持由非耗竭性T細胞表徵的IL-2產量、TNF-α產量、IFN-γ產量及顆粒酶B產量及/或維持在目標細胞存在下的離體殺傷能力，表明caTCR+CSR+c-Jun T細胞不經歷自活化及過早耗竭。 IV. 醫藥組合物 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

本文亦提供抗GPC3 caTCR加抗GPC3 CSR加c-Jun (「caTCR+CSR+c-Jun」)組合物(諸如醫藥組合物，在本文中亦被稱為調配物)，其包含免疫細胞(諸如T細胞)，在該免疫細胞表面上呈遞有根據本文所述之抗GPC3 caTCR中之任一者的抗GPC3 caTCR、根據本文所述之CSR中之任一者的抗GPC3 CSR及c-Jun多肽。在一些實施例中，caTCR+CSR+c-Jun免疫細胞組合物為醫藥組合物。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.

該組合物可包含：均質細胞群，其包含相同細胞類型之caTCR+CSR+c-Jun免疫細胞且表現相同抗GPC3 caTCR、抗GPC3 CSR及c-Jun；或異質細胞群，其包含有包含不同細胞類型之caTCR+CSR+c-Jun免疫細胞的複數個caTCR+CSR+c-Jun免疫細胞群，表現不同抗GPC3 caTCR、表現不同抗GPC3 CSR及/或表現不同c-Jun多肽(例如野生型或突變型c-Jun多肽)。該組合物可進一步包含不為caTCR+CSR+c-Jun免疫細胞之細胞。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.

在組合物製備期間的各個時刻，可能需要或宜低溫保存細胞。術語「冷凍(frozen/freezing)」及「低溫保存(cryopreserved/cryopreserving)」可互換使用。冷凍包括冷凍乾燥。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.

如一般技術者所理解，冷凍細胞可具破壞性(參見Mazur, P., 1977, Cryobiology14:251-272)，但存在大量可供防止此類破壞用之程序。舉例而言，可藉由(a)使用低溫保護劑、(b)控制冷凍速率及/或(c)在可使降解反應降到最低的足夠低的溫度下儲存來避免破壞。例示性低溫保護劑包括二甲亞碸(DMSO) (Lovelock及Bishop, 1959, Nature 183:1394-1395；Ashwood-Smith, 1961 , Nature 190:1204-1205)、甘油、聚乙烯吡咯啶酮(Rinfret, 1960, Ann. N.Y. Acad. Sci. 85:576)、聚乙二醇(Sloviter及Ravdin, 1962, Nature 196:548)、白蛋白、聚葡萄糖、蔗糖、乙二醇、i-赤藻糖醇、D-核糖醇、D-甘露糖醇(Rowe等人, 1962, Fed. Proc. 21 :157)、D-山梨糖醇、i-肌醇、D-乳糖、氯化膽鹼(Bender等人, 1960, J. Appl. Physiol. 15:520)、胺基酸(Phan及Bender, 1960, Exp. Cell Res. 20:651)、甲醇、乙醯胺、甘油單乙酸酯(Lovelock, 1954, Biochem. J. 56:265)及無機鹽(Phan及Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388；Phan及Bender, 1961, Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, Ilbery編, Butterworth, London, 第59頁)。在特定實施例中，可使用DMSO。添加血漿(例如，至20-25%之濃度)可強化DMSO之保護作用。在添加DMSO之後，細胞可保持在0℃下直至冷凍，因為1%之DMSO濃度在高於4℃之溫度下可具有毒性。 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.

在細胞之低溫保存中，緩慢的控制的冷卻速率可為關鍵的且不同低溫保護劑(Rapatz等人, 1968, Cryobiology5(1): 18-25)及不同細胞類型具有不同的最佳冷卻速率(關於冷卻速度對幹細胞之存活率及對於其移植潛力的影響，參見例如Rowe及Rinfret, 1962, Blood20:636；Rowe, 1966, Cryobiology3(1):12-18；Lewis等人, 1967, Transfusion7(1):17-32；及Mazur, 1970, Science168:939-949)。水變成冰之融化階段的熱量應該極少。冷卻程序可藉由使用例如可程式化冷凍裝置或甲醇浴液程序進行。可程式化冷凍設備允許測定最佳冷卻速率且促成標準可再現冷卻。 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.

在特定實施例中，經DMSO處理之細胞可於冰上預先冷卻且轉移至含有經冷藏之甲醇的托盤中，其又置放於機械製冷機(例如，Harris或Revco)中置於-80℃下。甲醇浴液及樣品之熱電偶量測指示1℃至3℃/分鐘之冷卻速率可為較佳的。在至少兩個小時之後，樣本可達到-80℃之溫度且可直接置放於液氮(-196℃)中。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).

在徹底冷凍之後，細胞可迅速轉移至長期低溫儲存容器中。在一較佳實施例中，樣品可低溫儲存於液氮(-196℃)或蒸氣(-1℃)中。高效液氮冰箱之可用性有助於此類儲存。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.

關於操控、低溫保存及長期儲存細胞之其他考慮因素及程序可見於以下例示性參考文獻中：美國專利第4,199,022號、第3,753,357號及第4,559,298號；Gorin, 1986, Clinics In Haematology 15(1):19-48；Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, 1968年7月22-26日, International Atomic Energy Agency, Vienna, 第107-186頁；Livesey及Linner, 1987, Nature 327:255；Linner等人, 1986, J. Histochem. Cytochem. 34(9):1 123-1 135；Simione, 1992, J. Parenter. Sci. Technol. 46(6):226-32。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.

在低溫保存之後，可解凍冷凍細胞以根據一般技術者已知之方法使用。冷凍細胞較佳快速解凍且在解凍後立刻冷藏。在特定實施例中，含有冷凍細胞之小瓶可浸入溫水浴中直至其頸部；平緩旋轉將確保細胞懸浮液隨著解凍發生混合且提高自溫水至內部冰塊的熱傳遞。一旦冰完全融化，即可立刻將小瓶置放於冰上。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.

在特定實施例中，可在解凍期間使用防止細胞凝集的方法。例示性方法包括：在冷凍之前及/或之後添加脫氧核糖核酸酶(Spitzer等人, 1980, Cancer 45:3075-3085)、低分子量聚葡萄糖及檸檬酸鹽、羥乙基澱粉(Stiff等人, 1983, Cryobiology 20:17-24)等。如一般技術者所理解，若使用對人類有毒的低溫保護劑，則其應在治療用途之前移除。DMSO無嚴重毒性。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.

例示性載劑及細胞投與模式描述於美國專利公開案第2010/0183564號第14-15頁。其他醫藥載劑描述於Remington: The Science and Practice of Pharmacy, 第21版, David B. Troy編, Lippicott Williams & Wilkins (2005)中。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).

在特定實施例中，細胞可自培養基收集，且洗滌並於載劑中濃縮至治療有效量。例示性載劑包括鹽水、緩衝鹽水、生理鹽水、水、漢克氏溶液(Hanks' solution)、林格氏溶液(Ringer's solution)、Nonnosol-R (Abbott Labs)、Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL)、甘油、乙醇及其組合。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.

在特定實施例中，載劑可補充有人血清白蛋白(HSA)或其他人類血清組分或胎牛血清。在特定實施例中，輸注載劑包括含5% HAS或右旋糖之緩衝鹽水。其他等張劑包括多羥基糖醇，包括三元醇或高級糖醇，諸如甘油、赤藻糖醇、***糖醇、木糖醇、山梨糖醇或甘露糖醇。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.

載劑可包括緩衝劑，諸如檸檬酸鹽緩衝劑、丁二酸鹽緩衝劑、酒石酸鹽緩衝劑、反丁烯二酸鹽緩衝劑、葡糖酸鹽緩衝劑、草酸鹽緩衝劑、乳酸鹽緩衝劑、乙酸鹽緩衝劑、磷酸鹽緩衝劑、組胺酸緩衝劑及/或三甲胺鹽。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.

穩定劑係指寬泛的一類賦形劑，其功能範圍可介於增積劑至有助於防止細胞黏附於容器壁上的添加劑之間。典型的穩定劑可包括多羥基糖醇；胺基酸，諸如精胺酸、離胺酸、甘胺酸、麩醯胺酸、天冬醯胺、組胺酸、丙胺酸、鳥胺酸、L-白胺酸、2-***酸、麩胺酸及蘇胺酸；有機糖或糖醇，諸如乳糖、海藻糖、水蘇糖、甘露糖醇、山梨糖醇、木糖醇、核糖醇、肌肉肌醇、半乳糖醇、甘油及環醇，諸如肌醇；PEG；胺基酸聚合物；含硫還原劑，諸如脲、麩胱甘肽、硫辛酸、巰乙酸鈉、硫代甘油、α-單硫代甘油及硫代硫酸鈉；低分子量多肽(亦即，＜10個殘基)；蛋白質，諸如HSA、牛血清白蛋白、明膠或免疫球蛋白；親水性聚合物，諸如聚乙烯吡咯啶酮；單醣，諸如木糖、甘露糖、果糖及葡萄糖；雙醣，諸如乳糖、麥芽糖及蔗糖；三醣，諸如棉子糖；及多醣，諸如聚葡萄糖。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.

在需要時或在有利時，組合物可包括諸如利多卡因(lidocaine)之局部麻醉劑以減輕注射部位之疼痛。Where necessary or advantageous, the composition may include a local anesthetic such as lidocaine to lessen pain at the site of the injection.

例示性防腐劑包括苯酚、苯甲醇、間甲酚、對羥基苯甲酸甲酯、對羥基苯甲酸丙酯、氯化十八烷基二甲基苯甲銨、鹵化苯甲烴銨、氯化六羥季銨、對羥基苯甲酸烷基酯(諸如對羥基苯甲酸甲酯或丙酯)、兒茶酚、間苯二酚、環己醇及3-戊醇。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.

組合物內之細胞的治療有效量可為大於10 2個細胞、大於10 3個細胞、大於10 4個細胞、大於10 5個細胞、大於10 6個細胞、大於10 7個細胞、大於10 8個細胞、大於10 9個細胞、大於10 10個細胞或大於10 11個細胞。 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.

在本文所揭示之組合物及調配物中，細胞之體積一般為一公升或更小、500 ml或更小、250 ml或更小、或100 ml或更小。因此，所投與之細胞密度通常為大於10 4個細胞/毫升、10 7個細胞/毫升或10 8個細胞/毫升。 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.

本文亦提供核酸組合物(諸如醫藥組合物，在本文中亦被稱為調配物)，其包含編碼本文所述之抗GPC3 caTCR、抗GPC3 CSR及/或c-Jun多肽的核酸中之任一者。在一些實施例中，核酸組合物為醫藥組合物。在一些實施例中，核酸組合物進一步包含以下中之任一者：等張劑、賦形劑、稀釋劑、增稠劑、穩定劑、緩衝劑及/或防腐劑；及/或含水媒劑，諸如純化水、糖水溶液、緩衝溶液、生理鹽水、聚合物水溶液或不含核糖核酸酶之水。待添加之此等添加劑及含水媒劑的量可根據核酸組合物之使用形式適當地選擇。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.

本文所揭示之組合物及調配物可經製備用於藉由例如注射、輸注、灌注或灌洗投與。組合物及調配物可進一步經調配用於骨髓、靜脈內、皮內、動脈內、結節內、***內、腹膜內、病灶內、***內、***內、直腸內、局部、鞘內、瘤內、肌肉內、囊泡內及/或皮下注射。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.

用於活體內投藥之調配物必須為無菌的。此要求容易藉由例如經由無菌過濾膜過濾實現。Formulations for intravenous administration must be sterile. This requirement is readily achieved, for example, by filtering through sterile filter membranes.

在一些實施例中，經工程改造之免疫細胞可自培養基收集，且洗滌且濃縮成呈治療有效量之載劑。例示性載劑包括鹽水、緩衝鹽水(例如磷酸鹽緩衝鹽水)、生理鹽水、水、漢克氏溶液(Hanks' solution)、林格氏溶液(Ringer's solution)、Nonnosol-R (Abbott Labs)、Plasma-Lyte A(R) (Baxter Laboratories, Inc., Morton Grove, IL)、甘油、乙醇及其組合。較佳地，載劑為等張的。在一些實施例中，載劑可補充有諸如人類血清白蛋白(HSA)或其他人類血清組分，5%葡萄糖或右旋糖的成分。亦可包括其他等張劑，包括多羥基糖醇，包括三元或更高級糖醇，諸如甘油、赤藻糖醇、***糖醇、木糖醇、山梨糖醇或甘露糖醇。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.

醫藥免疫細胞組合物(諸如醫藥T細胞組合物)可以治療有效量向癌症患者全身性(例如經由靜脈內或門靜脈注射)或局部(例如經由瘤內注射)投與。在一些實施例中，組合物(諸如靶向GPC3之組合物)用於治療患有肝細胞癌(HCC)、胃癌、胰臟癌或生殖系統癌症的患者(參見例如Wang及Wang, Canadian J Gastroent Hep.(2018) art. 9049252)。如本文所用，術語「治療(treatment)」或「治療(treating)」係指用於在經治療個體中獲得有益或期望結果之方法。此類結果包括但不限於緩解疾病(例如HCC)所引起之一或多個症狀、縮小疾病範圍(例如，減小腫瘤體積)、穩定疾病(例如，預防或延緩疾病惡化)、預防或延緩疾病擴散(例如轉移)、預防或延緩疾病再現或復發，改善疾病狀態、使得疾病緩解(部分或完全)、減少治療疾病所需的一或多種其他藥劑之劑量、改善生命品質、恢復體重及/或延長存活期(例如總存活期或無惡化存活期)。 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).

治療有效量之組合物係指足以實現所需臨床指標之經工程改造之免疫細胞(諸如T細胞)的數目。在一些實施例中，治療有效量含有超過10 4、10 5、10 6、10 7、10 8或10 9個經工程改造之細胞。 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.

在一些實施例中，醫藥組合物包含有效治療或預防疾病或病狀之量的細胞，諸如治療有效量或預防有效量。在一些實施例中，治療或預防功效藉由定期評估所治療之個體來監測。對於經數天或更長時間之重複投與，視病狀而定，重複治療直至出現疾病症狀之所需抑制為止。然而，其他給藥方案可適用且可確定。所需劑量可藉由單次推注投與組合物、藉由多次推注投與組合物或藉由連續輸注投與組合物來遞送。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.

在某些實施例中，個體被投與的範圍為約一百萬至約1000億個細胞，諸如100萬至約500億個細胞(例如約500萬個細胞、約2500萬個細胞、約5億個細胞、約10億個細胞、約50億個細胞、約200億個細胞、約300億個細胞、約400億個細胞或由以上各值中之任何兩者限定之範圍)，諸如約1000萬至約1000億個細胞(例如約2000萬個細胞、約3000萬個細胞、約4000萬個細胞、約6000萬個細胞、約7000萬個細胞、約8000萬個細胞、約9000萬個細胞、約100億個細胞、約250億個細胞、約500億個細胞、約750億個細胞、約900億個細胞或由以上各值中之任何兩者限定之範圍)，且在一些情況下，約1億個細胞至約500億個細胞(例如約1.2億個細胞、約2.5億個細胞、約3.5億個細胞、約4.5億個細胞、約6.5億個細胞、約8億個細胞、約9億個細胞、約30億個細胞、約300億個細胞、約450億個細胞)或此等範圍之間的任何值。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.

在一些實施例中，細胞及組合物係使用標準投藥技術、調配物及/或裝置來投與。提供調配物及用於儲存及投與組合物之裝置，諸如注射器及小瓶。投與可為自體或同種異體。舉例而言，免疫反應性細胞或祖細胞可自一位個體獲得且向同一個體或不同的相容個體投與。源於周邊血液之免疫反應性細胞或其後代(例如活體內、離體或活體外來源)可經由局域化注射進行投與，包括導管投藥、全身性注射、局域化注射、靜脈內注射或非經腸投藥。當投與本發明之治療性組合物(例如含有經基因修飾之細胞的醫藥組合物)時，其通常調配成單位劑量可注射形式(溶液、懸浮液、乳液)。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).

在一個態樣中，本發明提供醫藥組合物，其包含用於表現抗GPC3 caTCR、抗GPC3 CSR及c-Jun的核酸分子。核酸分子可以如上所述，例如上述病毒載體(例如，慢病毒載體)。醫藥組合物可離體用於工程改造T或祖細胞，接著將其引入患者中。醫藥組合物包含基因體包含抗GPC3 caTCR、抗GPC3 CSR及c-Jun之表現卡匣的核酸分子或重組病毒以及醫藥學上可接受之載劑，諸如緩衝溶液，其視情況包含其他試劑，諸如防腐劑、穩定劑及其類似試劑。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.

醫藥組合物可以提供為製品，諸如套組，其包括包含生物材料(細胞或核酸分子或重組病毒)之小瓶(例如，單劑量小瓶)及視情況存在之使用說明書。 V. 治療方法 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

本發明之表現構築體及/或組合物(諸如醫藥組合物)可被投與個體(例如哺乳動物，諸如人類)以治療涉及GPC3表現異常高的疾病及/或病症(在本文中亦稱為「GPC3陽性」疾病或病症)，包括例如癌症(諸如肝細胞癌(HCC))。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)).

因此，在一些實施例中，本申請案提供一種治療有需要之個體之GPC3陽性疾病的方法，其包含向個體投與有效量之包含一或多個表現抗GPC3 caTCR、抗GPC3 CSR及c-Jun多肽之免疫細胞的組合物(諸如醫藥組合物)。在一些實施例中，抗GPC3 caTCR為本文所提供之抗GPC3 caTCR中之任一者。在一些實施例中，抗GPC3 CSR為本文所提供之抗GPC3 CSR中的任一者。在一些實施例中，c-Jun多肽為本文所描述之c-Jun多肽中之任一者。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.

在一些實施例中，本文提供一種治療有需要之個體之癌症的方法，其包含向個體投與有效量之包含一或多個表現抗GPC3 caTCR、抗GPC3 CSR及c-Jun多肽(諸如本文所述之抗GPC3 caTCR、抗GPC3 CSR及c-Jun多肽中之任一者)之免疫細胞的組合物(諸如醫藥組合物)。在一些實施例中，癌症係選自例如由以下組成之群：HCC、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。在一些實施例中，癌症為HCC。在一些實施例中，癌症為HCC且治療包含預防癌症擴散，例如抑制(諸如預防)癌症轉移。在一些實施例中，癌症為轉移HCC。在一些實施例中，個體為人類。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.

在一些實施例中，本文提供一種治療有需要之個體之GPC3陽性疾病的方法，其包含向個體投與有效量之包含表現以下之一或多種免疫細胞的組合物(諸如醫藥組合物)：一或多種表現構築體，該等表現構築體包含用於表現以下之一或多個表現卡匣：a)抗GPC3 caTCR，其包含：i)特異性結合GPC3的抗原結合模組；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，抗GPC3 caTCR之TCRM源於人類γ/δ TCR。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，抗GPC3 CSR之跨膜模組包含源於CD30 (例如人類CD30)之跨膜域。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組源於CD30 (例如人類CD30)。在一些實施例中，人類c-Jun多肽為野生型人類c-Jun多肽。在一些實施例中，癌症係選自例如由以下組成之群：HCC、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。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.

在一些實施例中，本文提供一種治療有需要之個體之GPC3陽性疾病的方法，其包含向個體投與有效量之包含表現以下之一或多種免疫細胞的組合物(諸如醫藥組合物)：一或多種表現構築體，該等表現構築體包含用於表現以下之一或多個表現卡匣：a)抗GPC3 caTCR，其包含：i)抗原結合模組，該抗原結合模組包含：(a)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及(b)包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)配位體結合模組，該配位體結合模組包含：(a)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及(b)包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；ii)跨膜模組；及iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，抗GPC3 caTCR之抗原結合模組包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。在一些實施例中，caTCR為雜二聚體，其包含有包含SEQ ID NO: 30之胺基酸序列的第一多肽鏈及包含SEQ ID NO: 31之胺基酸序列的第二多肽鏈。在一些實施例中，caTCR為雜二聚體，其包含有包含SEQ ID NO: 10之胺基酸序列的第一多肽鏈及包含SEQ ID NO: 5之胺基酸序列的第二多肽鏈。在一些實施例中，抗GPC3 caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中第一與第二穩定域彼此具有使抗GPC3 caTCR穩定的結合親和力。在一些實施例中，抗GPC3 CSR之配位體結合模組包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，抗GPC3 CSR之抗GPC3配位體結合模組包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，抗GPC3 CSR之跨膜模組包含源於CD30 (例如人類CD30)之跨膜域。在一些實施例中，抗GPC3 CSR之協同刺激免疫細胞信號傳導模組源於CD30 (例如人類CD30)。在一些實施例中，抗GPC3 CSR包含SEQ ID NO: 44之胺基酸序列。在一些實施例中，抗GPC3 CSR包含SEQ ID NO: 16之胺基酸序列。在一些實施例中，人類c-Jun多肽為野生型人類c-Jun。在一些實施例中，人類c-Jun多肽包含SEQ ID NO: 1之胺基酸序列。在一些實施例中，癌症係選自例如由以下組成之群：HCC、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。 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.

在一些實施例中，本文提供一種治療有需要之個體之GPC3陽性疾病的方法，其包含向個體投與有效量之包含表現以下之一或多種免疫細胞的組合物(諸如醫藥組合物)：一或多種表現構築體，該等表現構築體包含用於表現以下之一或多個表現卡匣：a)抗GPC3 caTCR，其包含：i)包含特異性結合GPC3之Fab的抗原結合模組；及ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集；b)抗GPC3 CSR，其包含：i)包含能夠與GPC3結合或相互作用之scFv的配位體結合模組；ii)源於CD30之跨膜模組；及iii)源於CD30的能夠向免疫細胞提供協同刺激信號的協同刺激免疫細胞信號傳導模組，其中CSR缺乏功能性初級免疫細胞信號傳導域；及c)人類c-Jun多肽。在一些實施例中，Fab包含有包含SEQ ID NO: 28之胺基酸序列的重鏈可變域(V H)及包含SEQ ID NO: 29之胺基酸序列的輕鏈可變域(V L)。在一些實施例中，該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L。在一些實施例中，該scFv包含SEQ ID NO: 33之胺基酸序列。在一些實施例中，抗GPC3 CSR包含有包含SEQ ID NO: 44之胺基酸序列的CD30之片段。在一些實施例中，人類c-Jun多肽包含SEQ ID NO: 1之胺基酸序列。在一些實施例中，癌症係選自例如由以下組成之群：HCC、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。 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.

在一些實施例中，本文提供一種在有需要之個體中治療GPC3陽性疾病之方法，其包含向個體投與有效量之包含一或多種表現多順反子表現構築體之免疫細胞的組合物(諸如醫藥組合物)，該多順反子表現構築體包含用於表現以下之表現卡匣：a)抗GPC3 caTCR，其包含：i)特異性結合磷脂醯肌醇蛋白聚糖3 (GPC3)的抗原結合模組；及ii)衍生自人類γ/δ TCR之TCR模組(TCRM)；b)抗GPC3 CSR，其包含：i)能夠與GPC3結合或相互作用的配位體結合模組；ii)跨膜模組；及iii)衍生自人類CD30之胞內域的協同刺激免疫細胞信號傳導模組；及c)人類c-Jun多肽。在一些實施例中，表現卡匣包含SEQ ID NO: 1之編碼序列、SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33之編碼序列。在一些實施例中，編碼序列在框內藉由2A編碼序列或藉由內部核糖體進入位點(IRES)分開。在一些實施例中，表現卡匣包含組成性或可誘導型啟動子。在一些實施例中，啟動子為EF-1α啟動子。在一些實施例中，構築體為病毒載體(諸如慢病毒載體)。在一些實施例中，癌症係選自例如由以下組成之群：HCC、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。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.

為了能更好地理解本發明，闡述以下實例。此等實例僅為實現說明之目的且不應解釋為以任何方式限制本發明之範疇。 實例 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

參考以下實例將更充分理解本發明。然而，其不應解釋為限制本發明之範疇。應瞭解，本文所描述之實例及實施例僅出於說明之目的，且根據其之各種修改或變化將由熟習此項技術者提出且包括在本申請案之精神及範圍以及所附申請專利範圍之範疇內。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.

在以下實例1-3中描述之研究中，自Caliper及美國典型培養物保藏中心(American Type Culture Collection)獲得細胞株HepG2-luc (Caliper，源於ATCC HB-8065)及Hep3B (ATCC HB-8064)。HepG2及Hep3B皆為表現磷脂醯肌醇蛋白聚糖-3 (GPC3)的肝細胞癌(HCC)細胞株。在37℃/5% CO 2下，使細胞株在補充有10%胎牛血清(FBS)及2 mM麩醯胺酸之RPMI-1640 (Gibco)或DMEM (Thermo Fisher)中培養。 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 .

構築兩種慢病毒載體。第一種在本文中被稱為「GPC3-caTCR + GPC3-CD30-CSR」或「構築體1」，含有三順反子表現卡匣，該三順反子表現卡匣編碼GPC3特異性caTCR (SEQ ID NO: 30及31)及包含源於CD30之協同刺激域的GPC3特異性CSR (亦即GPC3-CD30-CSR) (SEQ ID NO: 16)。第二種載體在本文中被稱為「c-Jun + GPC3-caTCR + GPC3-CD30-CSR」或「構築體2」，含有四順反子表現卡匣，該四順反子表現卡匣編碼GPC3特異性caTCR (SEQ ID NO: 30及31)、GPC3-CD30-CSR (SEQ ID NO: 16)及人類c-Jun (SEQ ID NO: 1)。在各載體中，表現卡匣係在EF-1α啟動子之轉錄控制下，且不同多肽之編碼序列在框內藉由自裂解肽(F2A、T2A或P2A，如 圖 1A中所示)之編碼序列連接。藉由使用已知的慢病毒生產方案及封裝系統，用編碼構築體之載體轉染293T細胞，產生重組病毒。 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.

初代人類T細胞在用CD3/CD28珠粒(Dynabeads®, Invitrogen)在100 U/mL之介白素-2 (IL-2)存在下進行一天的刺激之後用於慢病毒轉導。將經濃縮慢病毒施加至用Retronectin® (Takara)塗佈之6孔盤中之T細胞且培育96小時。在第9天使用PE結合之抗人類F(ab')2抗體，藉由流動式細胞測量術評估轉導效率。使用BD FACSCanto II™收集流動式細胞測量術資料且使用FlowJo套裝軟體分析。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.

對於活體內研究，測試兩種肝癌模型(亦即HepG2-luc (研究1及研究2)及Hep3B模型(研究3))中表現構築體1或構築體2之初代人類T細胞之抗腫瘤活性。將表現螢光素酶之HepG2 (HepG2-luc)細胞或Hep3B細胞皮下(s.c.)植入NSG-MHC I/II DKO小鼠之右側腹(描述於 圖 1B中)。當腫瘤尺寸達到約150 mm 3(腫瘤細胞植入之後20天)時，向小鼠靜脈內(i.v.)注射經修飾T細胞。 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.

T細胞輸注對小鼠造成的健康影響係藉由監測動物之全身外觀、體重及不良反應之其他臨床徵象(包括體溫降低、呼吸困難及後肢麻痺/虛弱)來評估。另外，在T細胞給藥後第14天量測周邊血液T細胞計數。 實例 1 ： 活體外長期目標接合分析中過度表現 c-Jun 之抗 GPC3 caTCR + 抗 GPC3 CSR T 細胞之表徵 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

此實例描述c-Jun過度表現對人類T細胞之細胞毒性的作用，該等人類T細胞經工程改造以表現抗GPC3 caTCR及包含源於CD30之協同刺激域的抗GPC3 CSR (亦即GPC3-CD30-CSR)。具體言之，此實例展示，c-Jun過度表現促進表現抗GPC3 caTCR + 抗GPC3 CSR之T細胞的長期目標細胞殺傷能力。就長期T細胞耗竭而言，表現抗GPC3 caTCR + 抗GPC3 CSR之T細胞在存在或不存在c-Jun過度表現之情況下表現相當。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.

自健康人類供體分離之初代T細胞用慢病毒構築體1 (亦即GPC3-caTCR + GPC3-CD30-CSR)或構築體2 (亦即c-Jun + GPC3-caTCR + GPC3-CD30-CSR)轉導7-9天。藉由細胞Cytox 96®非放射性細胞毒性分析(Promega)來分析腫瘤細胞毒性，該分析定量地量測乳酸去氫酶(LDH)，一種在細胞溶解後釋放之穩定細胞溶質酶。CD3+ T細胞使用EasySep™人類T細胞分離套組(StemCell Technologies)由富含PBMC的全血製備，該套組不利耗乏表現CD14、CD16、CD19、CD20、CD36、CD56、CD66b、CD123及血型糖蛋白A之細胞。人類T細胞係根據製造商方案，用CD3/CD28 Dynabeads® (Invitrogen)活化及擴增。活化T細胞(ATC)在具有10% FBS加100 U/mL IL*2之RPMI-1640培養基中培養及維持，且在第7-14天使用。效應細胞藉由基於人類F(ab')2染色之caTCR表現(caTCR +)正規化(使用未轉導/經模擬轉導之T細胞將所有效應細胞樣品調整至T細胞總數當中相同百分比的受體 +(caTCR +)細胞)。 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).

將T細胞及目標細胞以1:1之起始效應細胞與目標細胞比率(E:T比)共培養。特定言之，在各孔中，在無細胞介素的情況下，將50,000個caTCR +T細胞與50,000個HepG2細胞於RPMI-1640及10% FBS中一起培育。為評估T細胞之長期殺傷潛力，該等細胞每7天每孔用100,000個HepG2細胞進行再攻擊。在各目標細胞接合之後，定量剩餘目標細胞及caTCR +T細胞之數目。 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.

長期殺傷(由剩餘目標細胞之量及剩餘T細胞數目表示)之比較展示於 表 3中。表現構築體2之T細胞經由比表現構築體1之T細胞多的再攻擊清除目標細胞，表明c-Jun促進抗GPC3 caTCR +抗GPC3 CSR +T細胞之長期目標細胞殺傷能力。 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.

在各目標細胞接合之後，藉由流動式細胞測量術分析CD4或CD8單一陽性caTCR +T細胞亞群。 表 3中之資料表明c-Jun過度表現產生較高百分比之CD4 +T細胞但較低百分比之CD8 +T細胞，表明c-Jun表現促進CD4 +T細胞優先擴增。 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.

為確定在長期殺傷分析期間用構築體1或構築體2轉導是否對T細胞分化具有影響，藉由流動式細胞測量術分析CD8 +受體 +細胞之CCR7及CD45RA之表現。初始T細胞由CCR7 +及CD45RA +表徵，而記憶T細胞由CCR7 +及CD45RA -表徵。如 表 3中所示，CCR7之表現量及記憶T細胞及初始T細胞之數目在表現構築體1之T細胞與表現構築體2之T細胞之間相當。此等結果指示c-Jun不影響抗GPC3 caTCR +抗GPC3 CSR +記憶T細胞之分化。 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.

為在長期殺傷分析期間檢查在經構築體1或構築體2轉導之T細胞上表現之耗竭標記物的含量，製備CD3 +T細胞且如上文所描述用CD3/CD28 Dynabeads®使細胞活化。將T細胞用慢病毒構築體1或構築體2轉導7-9天。效應細胞藉由基於人類F(ab')2染色之caTCR +正規化。如上所述培育T細胞且用HepG2細胞進行再攻擊。在各目標細胞接合之後的選定日藉由流動式細胞測量術分析caTCR +CD8 +T細胞及caTCR +CD4 +T細胞上耗竭標記物PD-1、LAG-3及TIM-3 (BioLegend®)之表現量。PD-1、LAG-3及TIM-3為隨著T細胞失去功能，在T細胞上積聚之抑制受體；因此，此等分子用作T細胞耗竭之標記物。 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.

如 表 3中所示，兩組T細胞顯示類似含量之耗竭標記物。此等結果證實，就長期耗竭而言，抗GPC3 caTCR +抗GPC3 CSR +T細胞在存在或不存在c-Jun過度表現之情況下的表現相當。 表 3. 活體外長期目標接合結果 比較特徵    構築體1 構築體2 CD3 +T細胞    相當 HepG2細胞清除       較高 受體 +T細胞    相當 受體 +T中之CD8 +T細胞    較高    受體 +T中之CD4 +T細胞       較高 CD8 +受體 +T中之CCR7表現    相當 CD8 +受體 +T中之初始T細胞    相當 CD8 +受體 +T中之中樞記憶T    相當                      PD1 CD8 +受體 + 相當 CD4 +受體 + 相當 LAG3 CD8 +受體 + 相當 CD4 +受體 + 相當 TIM3 CD8 +受體 + 相當 CD4 +受體 + 相當 實例 2 ： HepG2-luc 小鼠模型再攻擊研究中過度表現 c-Jun 之抗 GPC3 caTCR + 抗 GPC3 CSR T 細胞的活體內功效 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

此實例展示，相比於表現抗GPC3 caTCR + 抗GPC3 CSR而無c-Jun的T細胞，表現抗GPC3 caTCR + 抗GPC3 CSR且c-Jun過度表現的T細胞增加活體內腫瘤殺傷。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.

如上文所描述，評估來自表現構築體1或構築體2之兩個不同供體之初代人類T細胞在HepG2-luc模型中之活體內細胞毒性效能。在此等研究中，對小鼠靜脈內注射以下中之一者：(1) 5×10 6個未轉導之供體匹配(模擬)初代人類T細胞，(2) 5×10 6個表現構築體1之初代人類T細胞，(3) 2×10 6個表現構築體1之初代人類T細胞，(4) 5×10 6個表現構築體2之初代人類T細胞，或(5) 2×10 6個表現構築體2之初代人類T細胞(n=10隻小鼠/組)。為模擬腫瘤復發，對所有處理組中之小鼠進行腫瘤再攻擊研究，其中在T細胞給藥後約第70天，在左側腹中皮下植入HepG2-luc細胞。 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.

在所有四個處理組(亦即，高劑量構築體1、低劑量構築體1、高劑量構築體2、低劑量構築體2)中，在T細胞注射後約第10天，腫瘤開始消退；約20天實現完全腫瘤消退( 圖 2)。在用表現構築體1及構築體2之T細胞處理之動物的原發腫瘤曲線之間未觀測到顯著差異。此等結果展示c-Jun過度表現不影響「GPC3-caTCR + GPC3-CD30-CSR」 T細胞之原發腫瘤殺傷潛力。 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.

有趣地，當動物經歷腫瘤再攻擊時，觀測到c-Jun之增強效應。表現構築體2之T細胞的表現顯著優於表現構築體1之T細胞，表明在腫瘤再攻擊研究中c-Jun提高「GPC3-caTCR + GPC3-CD30-CSR」 T細胞之腫瘤殺傷潛力( 圖 3)。另外，在一項研究中，注射較高劑量之表現構築體2之T細胞的小鼠具有較高周邊血液T細胞計數( 圖 4A及 圖 4B)。 實例 3 ： Hep3B 小鼠模型中過度表現 c-Jun 之抗 GPC3 + 抗 GPC3 CSR T 細胞的活體內功效 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

此實例展示，相比於表現抗GPC3 caTCR + 抗GPC3 CSR而無c-Jun的T細胞，表現抗GPC3 caTCR + 抗GPC3 CSR且c-Jun過度表現的T細胞增加了活體內T細胞擴增。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.

評估Hep3B模型中的表現構築體1或構築體2之初代人類T細胞之活體內細胞毒性效能。對小鼠靜脈內注射以下中之一者：(1) 2.5×10 6個未轉導之供體匹配(模擬)初代人類T細胞，(2) 2.5×10 6個表現構築體1之初代人類T細胞，(3) 1.5×10 6個表現構築體1之初代人類T細胞，(4) 2.5×10 6個表現構築體2之初代人類T細胞，或(5) 1.5×10 6個表現構築體2之初代人類T細胞(n=10隻小鼠/組)。如 圖 5中所示，在初始消退之後，在所有組中觀測到腫瘤再生。表現構築體2之T細胞以比表現構築體1之T細胞更高的程度抑制復發性腫瘤之尺寸。在2.5 M之較高劑量下，藉由腫瘤體積所量測的構築體1與2之腫瘤抑制作用之間的差異係不可辨別的。另外，兩種劑量之構築體2組中之小鼠具有較高數目之周邊血液T細胞，表明c-Jun積極地影響活體內抗GPC3 caTCR +抗GPC3 CSR +T細胞擴增( 圖 4C)。 實例 4 ： 活體外長期目標接合分析中表現各種抗 GPC3 caTCR + 抗 GPC3 CSR 之 T 細胞之表徵 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

此實例描述人類T細胞的細胞毒性，該人類T細胞經工程改造以表現除人類c-Jun多肽以外的各種包含不同序列之抗GPC3 caTCR (例如GPC3-caTCR)以及各種包含不同序列及源於人類CD30之協同刺激域的抗GPC3 CSR (亦即GPC3-CD30-CSR)。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).

自健康人類供體分離之初代T細胞經下表4中所描述之表現構築體組中之一者轉導。 表 4. 包含抗 GPC3 部分之不同組合的例示性表現構築體 構築體描述 抗GPC3 caTCR V H/V L (SEQ ID NO) 抗GPC3 CSR V H/V L (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 24 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 24 25 c-Jun+GPC3-1 caTCR+GPC3-2-CD30 CSR 24 25 26 27 c-Jun+GPC3-1 caTCR+GPC3-3-CD30 CSR 24 25 28 29 c-Jun+GPC3-1 caTCR+GPC3-1-CD30 CSR 24 25 24 25 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

將表現各組表現構築體之T細胞與HepG2細胞共培養，且分析細胞毒性及耗竭程度，如上文實例1中所描述。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.

構築體c-Jun+GPC3-3 caTCR+GPC3-2-CD30 CSR (表4)與表4中所描述之其他構築體相比展示最高的長期目標細胞殺傷能力及最低的耗竭程度。 實例 5 ： 表現抗 GPC3 caTCR 及不同的抗 GPC3 CSR 的 T 細胞之表徵 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

此實例表徵人類T細胞，該人類T細胞經工程改造以表現除人類c-Jun多肽以外的抗GPC3 caTCR (例如GPC3-caTCR)以及各種包含不同協同刺激片段(例如包含協同刺激域及跨膜域的協同刺激片段)的抗GPC3 CSR (例如GPC3-CSR)。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).

自健康人類供體分離之初代T細胞經下表5中所描述之表現構築體組中之一者轉導。各例示性GPC3-caTCR編碼GPC3-3之V H/V L序列(參見序列表)，且各例示性GPC3-CSR編碼GPC3-2之V H/V L序列(參見序列表)。 表 5. 包含有包含不同協同刺激片段之抗 GPC3-CSR 的例示性表現構築體 構築體描述 協同刺激(IC) 域 跨膜(TM) 域 抗GPC3 caTCR V H/V L (SEQ ID NO) 抗GPC3 CSR V H/V L (SEQ ID NO) V H V L V H V L c-Jun+GPC3-3 caTCR (無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 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

在實例5A-5G中執行且描述了表現表5中所描述之構築體的T細胞(例如表現相同抗GPC3-caTCR (例如GPC3-3 caTCR)及c-Jun，但具有包含不同協同刺激片段之不同抗GPC3-CSR的T細胞)的比較分析。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.

總體而言，實例5A-5G展示c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM-CD30 IC-CSR及c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR表現同樣良好且比其他抗GPC3-CSR好。 實例 5A ： 短期活體外癌細胞殺傷分析 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

表現表5中所描述之構築體中之各者的T細胞與HepG2細胞共培養，且如實例1中所描述，藉由Cytox 96®非放射性細胞毒性分析(Promega)來分析腫瘤細胞毒性，該分析定量地量測釋放至培養物上清液中之乳酸去氫酶(LDH)。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.

表現c-Jun+GPC3-3 caTCR+GPC3-2-CD30 TM-CD30 IC-CSR或c-Jun+GPC3-3 caTCR+GPC3-2-CD28 TM-CD30 IC-CSR之T細胞具有比表5中所概述之其他不具有CSR或包含其他協同刺激片段之CSR的構築體高的殺傷功效。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.

表現表5之構築體之各種T細胞的長期殺傷能力亦藉由量測在與HepG2細胞接合後自T細胞釋放之細胞介素的量/含量來測定。用Luminex Magpix技術使用BioRad Bio-Plex套組或藉由ELISA，定量16小時共培養之後上清液中細胞介素釋放之含量。具有較高細胞毒性效能之T細胞分泌高含量之與T細胞活性相關的細胞介素，諸如TNFα、GM-CSF、IFNγ及IL-2。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.

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞具有比無任何CSR之彼等T細胞高，且比含有不包含CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)的CSR的彼等T細胞高的或與之大約相同的殺傷功效(參見表5)。 實例 5B ：增殖潛力及持久性分析 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

經基因修飾之T細胞的增殖及持久性對於治療癌症時授受性T細胞轉移療法的成功而言至關重要。為分析特定CSR對於T細胞增殖及持久性之影響，將表現表5之構築體中之各者的T細胞用胞內染料5-(及-6)-羧基螢光素二乙酸丁二醯亞胺酯(CFSE)標記，且當T細胞在腫瘤細胞刺激下***時，觀測到染料被稀釋。藉由對指定日剩餘之CFSE陽性細胞之數目進行計數來量測T細胞之持久性。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.

使表現表5中所示之構築體中之各者的T細胞之血清饑餓隔夜且用CFSE使用CellTrace CFSE (Thermo Fisher)標記。50,000至100,000個T細胞以2:1之效應細胞與目標細胞比率(E:T比)培育，且當T細胞在指定日***時，使用流動式細胞測量術觀測CFSE染料之連續稀釋。T細胞總數目用FACS計數。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.

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞增殖的程度大於不具有任何CSR之對應CAR T細胞，且增殖的程度大於表現不包含CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)之CSR的對應CAR T細胞或與之大約相同。 實例 5C ： 多週接合後的活體外 T 細胞及腫瘤細胞計數 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

使用用於對目標細胞進行計數之基於FACS之分析來比較表現表5中所描述之構築體中之各者的T細胞之長期殺傷潛力。表現表5中所描述之構築體中之各者的T細胞在目標細胞接合後展示出相當的存活率。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.

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞相較於不具有CSR之對應CAR T細胞在與腫瘤細胞多次接合中持續較長時段且殺傷更多腫瘤細胞，且與含有不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)的CSR的CAR T細胞大約相同(若非更佳)。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).

使用用於計數T細胞及目標細胞之基於FACS之分析來比較表現表5中所描述之構築體之T細胞的長期存活率及目標細胞殺傷潛力。將T細胞共培養且如實例1中所述用目標細胞對其進行再攻擊。在各目標細胞接合之後的不同天數，用FACS定量剩餘目標細胞及總T細胞之數目。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.

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞相較於不具有任何CSR之對應T細胞在腫瘤目標細胞之多次接合中持續/存活較長時段且殺傷更多腫瘤細胞，且比表現不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)的CSR的對應T細胞存活得好及/或殺傷更多腫瘤細胞或者與之大約相同。 實例 5D ：活體外 T 細胞耗竭 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

為了檢查經表5中所述之構築體轉導之T細胞上表現的耗竭標記物含量，caTCR +CD8 +T細胞及caTCR +CD4 +T細胞上耗竭標記物TIGIT、PD-1、LAG-3及TIM-3 (BioLegend®)之表現量係在各目標細胞接合之後的選定天數藉由流動式細胞測量術分析，如實例1中所述。 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.

相較於不具有任何CSR之對應T細胞或表現不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)之CSR的T細胞，表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞減少了長期耗竭標記物。 實例 5E ：活體內細胞介素釋放 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

分別如實例2及實例3中所描述，評估表現表5之構築體中之各者的初代人類T細胞在HepG2-luc及Hep3B小鼠模型中的活體內細胞毒性效能。簡言之，為測定活體內細胞介素釋放水平，在投與T細胞之後16、24、48及72小時，分析關鍵細胞介素，包括與臨床細胞介素釋放症候群相關之細胞介素。使用BioRad Bio-Plex套組，藉由Luminex Magpix技術定量細胞介素含量。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.

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞比不具有任何CSR之對應T細胞或表現不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)的CSR的T細胞分泌更高含量之與T細胞活性相關的細胞介素，諸如TNFα、GM-CSF、IFNγ及IL-2。 實例 5F ： T 細胞亞群隨時間之分化 (CCR7/CD45RA) 及記憶 T 細胞定量 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

在兩個非依賴性流動式細胞測量分析中，表現表5中所描述之表現構築體之T細胞的增殖及存活率係在目標細胞接合前後量測。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.

對表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞的FACS分析展示比不具有任何CSR之T細胞或表現不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)的CSR的T細胞更高的T細胞分化標記物CCR7及CD45RA之表現量以及增加的記憶T細胞及初始T細胞之百分比。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).

表現c-Jun、抗GPC3 caTCR及抗GPC3-CD30-CSR的T細胞在目標刺激之後發展成且維持高量的記憶T細胞群，包括中樞記憶T細胞及效應記憶T細胞。為確定不同CSR對T細胞發展成且維持記憶T細胞之能力的影響，量測記憶T細胞標記物CCR7及CD45RA之細胞表面表現。如此項技術中已知，具有高CCR7表現量及低CD45RA表現量之T細胞被視為中樞記憶T細胞，具有低CCR7及低CD45RA表現量之T細胞被視為效應記憶T細胞，具有低CCR7及高CD45RA表現量之T細胞被視為效應T細胞，而具有高CCR7及高CD45RA之T細胞為初始T細胞，即為在靶向/抗原攻擊/識別之前的初始類型之T細胞(Mahnke等人, Eur J Immunol.43(11):2797-809, 2013)。回應於抗原遭遇，初始T細胞增殖且分化成效應細胞，其中大多數執行破壞目標之工作，且隨後死亡，而小的T細胞池最終發展成能儲存針對特異性目標之T細胞免疫性的長期記憶T細胞。在記憶T細胞當中，發現中樞記憶T細胞的壽命比效應記憶T細胞長，且能夠產生效應記憶T細胞，但反之則不然。因此，發展成且維持記憶T細胞，尤其是中樞記憶T細胞的能力係可能的成功T細胞療法之重要且所需特徵。 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.

表現c-Jun及抗GPC3 caTCR但無CSR之T細胞與HepG2細胞以2:1之E:T比(例如，在96孔盤之各孔中，100,000個受體 +T細胞及50,000個HepG2細胞)一起培育7天。細胞隨後每7天每孔用50,000-100,000個HepG2細胞再攻擊。表現不同CSR之其他組中之T細胞與目標細胞以1:2之E:T比(例如各孔中25,000受體 +T細胞及50,000 HepG2細胞)一起培育7天。細胞隨後每7天每孔用50,000-100,000個HepG2細胞再攻擊。 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.

在一些實驗中，將T細胞及HepG2細胞混合物在第四及第五次HepG2細胞接合之前以1:6稀釋，以避免由於顯著的T細胞擴增而致使T細胞過度擁擠，使得僅六分之一的先前剩餘細胞被50,000-100,000個HepG2細胞再攻擊。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.

在各HepG2細胞接合之後的選定日，來自各樣品的孔中之全部細胞混合物用針對CCR7及CD45RA之抗體染色且藉由流動式細胞測量術分析。對受體 +T細胞數目進行計數，且基於細胞的CCR7及CD45RA表現量將細胞分組為各種T細胞類型：中樞記憶T細胞(CD45RA -CCR7 +)、效應記憶T細胞(CD45RA -CCR7 -)、效應T細胞(CD45RA+ CCR7 -)及初代T細胞(CD45RA +CCR7 +)。計算受體 +T細胞總數目當中各種類型之T細胞的百分比。在一些實驗中，細胞亦用針對CD8或CD4之抗體染色以測定CD8 (細胞毒性T細胞)及CD4 (輔助性T細胞) T細胞類型之組合物。 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.

表現c-Jun、抗GPC3 caTCR及抗GPC3 CSR之T細胞的增殖及存活係在目標細胞接合前後量測。表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞在與HepG2細胞接合後能夠發展成且維持高數目及高百分比的中樞記憶T細胞，高於無任何CSR之T細胞或表現不具有CD30 IC域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)之CSR的T細胞。 實例 5G ： HepG2 或 Hep3B 小鼠模型中 T 細胞之活體內功效 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

在NSG小鼠中皮下植入約10 7個HepG2腫瘤細胞或Hep3B且使其形成150 mm 3實體腫瘤塊。將表現表5中所述之構築體中之各者的5×l0 6個T細胞靜脈內注射至攜帶腫瘤之小鼠中。在T細胞給藥後3週，處死小鼠且移除腫瘤，固定且切片至載片上。腫瘤切片用CD3抗體染色以觀察存在於實體腫瘤內之T細胞。對CD3 +細胞數的定量可用於對T細胞之腫瘤浸潤能力(T細胞/mm 2)進行評分。 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 ).

表現c-Jun、抗GPC3 caTCR及包含至少CD30 IC域之抗GPC3 CSR的T細胞具有比無任何CSR之T細胞或表現不具有CD30域但具有不同協同刺激分子IC域(例如CD28或4-1BB IC域)之CSR的T細胞高的活體內腫瘤浸潤/滲透率/含量(亦即，更高的T細胞數/mm 2)。 序列表 SEQ 描述 序列 1             人類c-Jun MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF 2             信號肽 METDTLLLWVLLLWVPGSTG 3             部分TCR γ QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 4             部分TCR δ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 5             caTCR - 2 多肽2 QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 6             GPC3-1 HC-CDR1 GFTFSSYA 7             GPC3-1 HC-CDR2 IYSGGSST 8             GPC3-1 HC-CDR3 ARTSYLNHGDY 9             GPC3-1 LC-CDR1 RSNIGSDY 10          caTCR - 2 多肽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 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSAAATGAPPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 17          GPC3-2 LC-CDR3 QVWDSSSDHYV 18          GPC3-3 HC-CDR1 GYTFASHG 19          GPC3-3 HC-CDR2 ISPYTGNT 20          GPC3-3 HC-CDR3 ARGKRTLASCFDY 21          GPC3-3 LC-CDR1 NIGSKS 22          CD8跨膜域 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC 23          GPC3-3 LC-CDR3 QVWDSSSDHV 24          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 多肽1 QVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 31          caTCR – 1 多肽2 QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 32          GPC3-1 scFv QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSS 33          GPC3-2 scFv QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSS 34          GPC3-3 scFv QSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVFGTGTKVTVLGSRGGGGSGGGGSGGGGSLEMAQVQLVQSGADVRKPGASVKVSCKASGYTFASHGISWVRQAPGQGLEWLGWISPYTGNTNYAQKFQGRVTMATDTSTSTAYMELRSLRSDDTAIYYCARGKRTLASCFDYWGQGTLVTVSS 35          TCRα恆定域 PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 36          TCRβ恆定域 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 37          TCRδ恆定域 SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 38          TCRγ恆定域 DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 39          TCRα跨膜域 ILLLKVAGFNLLMTLRLWSS 40          TCRβ跨膜域 TILYEILLGKATLYAVLVSALVL 41          TCRδ跨膜域 VLGLRMLFAKTVAVNFLLTAKLFFL 42          TCRγ跨膜域(與uniprot相同) YYMYLLLLLKSVVYFAIITCCLL 43          CD30協同刺激片段1 APPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 44          CD30協同刺激片段2 HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 45          CD28協同刺激片段1 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 46          CD28協同刺激片段2 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 47          4-1BB協同刺激片段1 PADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 48          4-1BB協同刺激片段2 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 49          CD30跨膜域 PVLDAGPVLFWVILVLVVVVGSSAFLLC 50          CD28跨膜域 FWVLVVVGGVLACYSLLVTVAFIIFWV 51          CD8跨膜域 IYIWAPLAGTCGVLLLSLVIT 52          c-Jun核苷酸序列#1 atgacagccaagatggaaaccacattctacgacgacgccctgaacgcctcattcctgccttctgagagcggaccttacggctacagcaatcctaagatcctgaaacagagcatgacccttaacctggctgatcctgttggaagcctgaaacctcacctgagagccaaaaacagcgacctgctcaccagccctgatgtgggcctgctgaagctggcctctccagagctggaacggctgatcatccagagcagcaacggccacatcacaaccacccctacccctacacaattcctgtgccctaagaacgtgaccgacgagcaggagggcttcgccgaaggctttgtgcgggccctggcagaactgcactctcagaacaccctgcctagcgtgacctccgccgcccagcctgtcaacggcgccggaatggtggcccctgccgtggcttctgtggccggcggcagcggcagcggcggattcagcgcctctctgcactctgagcctcctgtctacgccaatctgtctaatttcaaccccggagccctgtccagcggcggcggagctcctagctacggcgctgctggactggccttccccgcccagccccagcaacagcagcagcctccacaccacctgccccagcagatgcccgtgcagcaccctagactgcaggccctgaaggaagaaccccaaacagtgcctgagatgcctggcgagacacctccactgagccccatcgacatggaaagccaggagcggatcaaggccgagagaaagagaatgcggaacagaatcgccgctagcaagtgcagaaagcggaagctggaaagaatcgccagactggaagagaaggtgaagaccctgaaagcccaaaatagcgagctggccagcaccgccaacatgctgcgggaacaggtggcccagctgaagcagaaggtgatgaaccacgtgaactctggttgtcagctgatgctgacccagcagctccagaccttc 53          c-Jun核苷酸序列#2 atgacagccaagatggaaaccaccttctacgacgacgccctcaacgcctccttcctgccttctgagagcggtccttacggctacagcaaccccaagatcctgaagcaaagcatgaccctgaacctggccgaccccgttggctccctgaaacctcacctgagagccaaaaacagcgacctgctgaccagccctgatgtgggcctgctgaagctggcctctccagagctggaaagactgattatccagagcagcaacggccacatcaccacaacacctacccctacacagttcctgtgccctaagaacgtgactgatgagcaggagggctttgccgagggcttcgtgagagccctggctgagctgcattctcagaacaccctgcctagcgtgacctctgccgcccagcctgttaatggcgccggcatggtggcccctgccgtggcctctgtggccggaggcagcggcagcggcggattcagcgcctctctgcacagcgagccccccgtctacgccaacctgagcaatttcaaccctggcgccctgtccagcggcggcggcgccccttcatatggcgctgccggcctggccttccccgctcagccccagcagcagcaacagcctccacaccacctgccccagcagatgcccgtgcagcaccccagactgcaggccctgaaggaagaacctcagaccgtgcccgagatgcctggcgagacccctcctctgagccctatcgacatggaaagccaggagagaatcaaggccgagaggaagcggatgcggaacagaatcgccgccagcaagtgcagaaaaagaaagctggaacggatcgccagactggaggagaaggtgaagacactgaaagcccaaaattctgaactggcctctaccgccaatatgctgcgcgagcaggtggctcaactgaagcagaaggtgatgaaccacgtgaacagcggatgtcagctgatgctgacacagcagctgcagactttt 54          c-Jun核苷酸序列#3 atgaccgccaagatggaaaccaccttctacgacgacgccctgaacgccagctttctgccttctgagtctggcccctacggctacagcaaccccaagatcctgaagcagagcatgaccctgaacctggccgatcctgtgggcagcctgaaacctcacctgagagccaagaacagcgacctgctgacaagccctgatgtgggcctgctgaaactggcctctcctgagctggaacggctgatcatccagagcagcaacggccacatcaccaccacacctacaccaacacagtttctgtgccccaagaacgtgaccgacgagcaagagggattcgccgagggctttgttagagccctggccgaactgcacagccagaataccctgcctagcgtgacatctgccgctcagcctgttaatggcgccggaatggttgctcctgccgtggcttctgttgctggcggatctggatctggcggctttagcgcctctctgcactctgagcctccagtgtacgccaacctgagcaacttcaaccctggcgctcttagctctggtggcggagcaccttcttatggcgctgccggattggcctttcctgctcagcctcagcagcagcaacagcctcctcatcatctgccccagcagatgcctgtgcagcaccctagactgcaggccctgaaagaggaaccccagacagtccctgagatgcccggcgaaacacctcctctgagccccatcgacatggaaagccaagagcggatcaaggccgagcggaagcggatgagaaatagaatcgccgcctccaagtgccggaagaggaagctggaaagaatcgcccggctggaagagaaagtgaaaaccctgaaggcccagaactccgagctggcctctaccgccaacatgctgagagaacaggtggcccagctgaaacagaaagtcatgaaccacgtgaacagcggctgccagctgatgctgacacagcagctgcagaccttc 55          c-Jun核苷酸序列#4 atgactgccaaaatggagactacattctatgacgacgccctcaatgccagttttttgccgagtgaatccggcccctacggctattcaaaccctaagatcctcaagcaatcaatgaccctcaatcttgctgacccagttggctccctgaaaccccatctcagagctaaaaatagtgacctccttacttcccctgatgttggactcctcaaacttgcttctcccgaactcgaacgcttgatcattcaatcttccaacggccacatcacaacaacacccacacccacccagtttctttgcccaaaaaatgtcaccgatgaacaggaaggtttcgcggaaggattcgtccgcgcgctggccgaactgcactcccagaatacacttccttcagttacgtcagccgcccagccagtgaatggtgcgggaatggttgctcctgcggtcgcttctgtcgcagggggctccggttctggcggatttagcgcctctctgcattccgagccacctgtatatgctaatctttctaattttaaccccggagccttgtctagcggcggtggtgcccccagctacggtgctgcaggactcgccttcccagctcaacctcagcagcagcaacaacccccccatcaccttccccaacagatgccagtacaacatccaaggctccaggccctcaaagaggaaccacagacggtgcccgaaatgcctggcgaaactccaccactttcccctattgatatggaatcccaagagcgcatcaaggccgaaagaaagcgaatgcggaatagaatagcagcttcaaaatgtagaaaacggaaattggaacgaatcgcacggttggaagaaaaggtgaagaccttgaaagcccagaacagtgagctcgcctctaccgctaacatgctgcgcgagcaagtcgcacaacttaagcagaaggtgatgaaccatgtgaatagcggatgtcaacttatgctgactcaacagttgcaaaccttt 56          c-Jun核苷酸序列#5 atgaccgcgaaaatggagacaacattttacgatgatgcactgaacgcctcttttctgccaagtgaatccggcccctacggatactcaaaccctaagattctgaaacagtctatgactctcaacctggccgacccagttggcagtctgaagcctcatttgcgagccaagaatagtgatctgctgacctccccagacgtgggactgctgaaactcgcctcacctgaacttgagcgcttgattatacagtcatccaatgggcacatcacaacaacacctactcctacccagtttctgtgccccaaaaacgtcaccgatgagcaggagggattcgcggaaggctttgtgcgcgccctggctgaattgcatagtcagaacactcttcccagcgtaaccagcgccgcccaaccagtgaatggagccggtatggtggctcccgcggtggctagtgttgcgggggggtcaggctctggtgggttcagtgcttctcttcactctgaaccccctgtgtatgccaatctgtctaactttaaccctggggccctctcctctggtgggggtgcccccagctacggagcggccggcctggcctttcctgcccagcctcagcagcagcagcaaccccctcatcatcttccgcagcagatgccagtacagcatccacgcctgcaggctcttaaggaggagccccagacggtgcccgaaatgcccggggaaactccacccttgtcccccattgacatggagtcccaggagcggatcaaggctgaaagaaagaggatgcggaatcgcatcgcagcctctaaatgccgcaagcggaaacttgagaggatcgcgcggttggaggaaaaagtaaaaaccttgaaggcacagaactctgagctggcgagtactgccaacatgctcagagaacaagtcgcacagctgaagcagaaagtgatgaaccatgtgaacagcggttgtcagctgatgctgactcagcagctgcagaccttc 57          c-Jun核苷酸序列#6 atgaccgccaagatggagaccacattctacgatgacgctctgaacgcttcctttctgccttccgagtccggcccctacggctactccaatcccaagattctgaagcagagcatgacactgaatctggctgatcccgtgggatctctgaagcctcatctgagagccaagaattccgatctgctgacaagccccgacgtgggactgctcaaactggccagccccgaactggagaggctcattatccagagctccaacggccacatcaccacaacacctacccctacccagtttctctgtcccaagaacgtgacagacgagcaagagggatttgccgaaggcttcgtgagagccctcgccgaactgcatagccagaacacactgccttccgtgaccagcgctgctcaacccgtgaacggcgctggcatggtcgctcccgccgtcgccagcgtggctggaggaagcggatccggaggcttcagcgcttccctccacagcgaacctcccgtgtacgctaatctgagcaacttcaaccccggcgctctgagcagcggaggaggagctcctagctatggagctgccggactggcttttcccgcccagccccagcagcagcagcagcccccccatcatctgcctcagcagatgcccgtgcagcatcccagactccaagctctgaaggaggagcctcagaccgtccccgagatgcccggcgaaaccccccctctgtcccccatcgacatggaaagccaagagaggatcaaggccgagaggaagaggatgaggaatagaatcgccgccagcaagtgtagaaagaggaagctggagaggatcgccagactggaggagaaggtgaagaccctcaaggctcagaattccgagctggccagcacagccaacatgctgagagagcaagtggcccagctcaagcagaaggtgatgaaccacgtcaacagcggatgccagctgatgctcacccagcagctgcagaccttc 58          c-Jun核苷酸序列#7 atgaccgctaaaatggaaaccactttctatgacgatgccctgaacgcctccttccttccgtccgagtccggaccctacggatactcaaatcctaagatcctcaaacagtcgatgaccctcaacctggccgaccccgtgggatccctgaagccgcacttgcgcgccaagaactccgacctcctgacgagcccagacgtgggcctgctgaagctcgcatcacccgaacttgagcggttgatcattcagtcctccaacggacatatcaccaccactcccaccccaactcagtttctgtgtccgaagaacgtgaccgatgagcaagagggattcgccgagggattcgtgcgggccctggccgagctgcatagccagaacacccttccatccgtgacctcggcggctcagcctgtgaacggcgcgggaatggtcgcgcccgccgtggcctcggtggccgggggcagcggcagcgggggattttccgcgtcgctgcactccgagccgccggtgtacgccaacctgtcaaacttcaaccctggggccctgagctccggcggtggagcaccttcgtacggcgccgctggcctggcgttccccgcgcaaccacagcagcaacagcagccccctcaccacctcccccaacaaatgcctgtgcagcacccgaggctgcaggccctcaaggaagaaccccagactgtgccggaaatgccgggggagactccgccgctgtcccctatcgacatggaatcacaggaacgcattaaggcagagcggaagcgcatgcggaaccggattgccgcctccaagtgccgcaagagaaagctcgaaagaatcgccagattggaagaaaaggtcaagactctgaaggcccagaactctgagctggcatccaccgctaatatgctgagggaacaagtggcccagctgaaacagaaggtcatgaaccacgtcaacagcggttgccagctgatgctgacccagcaactccagacattc 59          c-Jun核苷酸序列#8 atgaccgccaagatggagaccaccttctacgacgacgccctgaacgccagcttcctgcccagcgagagcggaccctacggctactctaaccccaagatcctgaaacagagcatgacactgaatctggccgaccccgtgggcagcctgaagcctcaccttagagccaagaacagcgacctgctgaccagccccgacgtgggcctgctgaagctcgcctctccagagttagagagactgatcatccagtccagcaacggccacatcacaaccaccccaacccctacccagttcctgtgccccaagaacgtgaccgacgagcaggagggcttcgccgagggctttgtgagagccctggccgagttgcactctcagaacaccctgccctccgtgaccagcgccgctcaacctgtgaacggcgcaggaatggttgctcctgccgtggccagcgttgcaggcggatctggaagtggaggcttctccgcctcccttcacagcgagcctcccgtgtacgccaacctgagcaacttcaaccccggcgccctgagcagtggaggaggcgctcccagctatggagcagctggattagccttccccgcccagccacagcagcagcaacagcctccccaccacctgcctcagcaaatgcctgtgcagcaccctcggctgcaggcccttaaggaggagccccagaccgttcctgagatgcctggcgagacccctcccctgagccctatcgacatggagtcccaggagcggatcaaggccgagcggaagcggatgcggaaccggatcgctgcttccaagtgccggaagagaaagctggagagaatcgcccggctggaggagaaggtgaagaccctgaaggcccagaactccgagctggcctccaccgccaacatgctgcgggagcaggttgcacagctgaagcagaaggtcatgaaccacgtgaacagcggctgccagctgatgctgacccagcagctgcagaccttc 60          c-Jun核苷酸序列#9 atgacagcgaagatggagacaaccttctatgacgatgctcttaacgcctccttcctgccttccgaaagcgggccctacgggtactctaatcctaagatacttaagcaatcgatgactctcaacctcgctgacccggttggctcactgaaaccacacctgagagctaagaatagtgacctgctcactagtcccgatgtcgggcttctgaagctggcctctcccgagctggagaggcttatcatccaatcatcaaatggccacatcaccactaccccaacaccaactcaattcctttgccctaaaaacgtgaccgacgaacaggaaggcttcgccgagggttttgtccgggccttggccgagctgcattctcaaaatacactgccaagcgtcacttctgcggcgcagccggttaacggagcagggatggtggctcccgccgttgctagcgtggccggcggttccggctccggcggtttctctgcctccttgcattctgagccaccagtctacgcgaacctgtccaactttaatccgggggcgctgagtagcggaggcggcgcccctagctatggggcagctggactggccttcccggcacaaccccaacaacaacagcaaccgccacaccatcttcctcaacaaatgccagtgcaacatccacgcttacaagccctcaaggaggaaccccagaccgtgcctgagatgcccggcgaaaccccgccattgagccctattgacatggaaagtcaagagagaattaaggcagagcgcaagagaatgaggaaccggatcgcagcatctaagtgccgcaaacggaaattggagcggatcgctcgcttggaggagaaggtcaagactctcaaggcccagaactccgagcttgcgagcacagctaatatgctgcgcgagcaggtggcccagttaaaacaaaaggtcatgaaccatgtgaacagcggctgtcagctgatgcttacgcaacagctgcaaacctttggctccggtgcaacgaacttcagcctgctgaagcaggccggagatgttgaggaaaatccaggtccc 61          c-Jun核苷酸序列#10 atgacggccaaaatggagactacgttctacgatgacgcactcaacgcgtccttcctgccctctgagagtggaccctatggctactccaatccaaagatcctgaagcagtctatgaccctcaacctggcggacccggtgggctcccttaagccgcacttgcgcgccaagaactccgacctgctgacctcccctgatgtgggcctcctcaagctcgctagccctgaattggagaggctgatcatccagagctcaaatggccacatcaccaccacacctaccccaacccagttcctgtgcccaaaaaacgtgaccgacgagcaggagggcttcgcggagggcttcgtcagagctctggccgagctgcactcacagaacacgctcccttccgtgacctccgctgcccagccggtcaatggcgctggaatggtggctccggctgtggcctctgttgccggcggctccggctccggaggcttttcagcttctctgcattctgagcccccagtgtacgctaacctgagcaacttcaaccccggggcgctcagctccggtggcggtgccccgagctacggcgcggctgggctggcgttccccgctcagcctcagcagcaacagcaacctccccaccacctgccacagcagatgcctgtgcagcacccacgcctgcaggccttgaaggaggaacctcagactgtgccagagatgcccggcgagaccccacccctgtccccgattgacatggagagccaggagcgcatcaaggcagagcgcaagcgtatgcgcaaccgcatcgcggcctccaagtgccgaaagcgcaagctggagcggattgctcgcctggaggagaaggtgaagaccctgaaggcccagaattccgagctggcctcgaccgccaacatgctacgagaacaggtcgcgcagctgaaacagaaggtcatgaaccatgtcaacagcgggtgccagctgatgttgacccagcagcttcagaccttc 62          野生型Jun核苷酸序列 (GenBank登錄號NM_002228.4) *編碼區大寫 gctcagagttgcactgagtgtggctgaagcagcgaggcgggagtggaggtgcgcggagtcaggcagacagacagacacagccagccagccaggtcggcagtatagtccgaactgcaaatcttattttcttttcaccttctctctaactgcccagagctagcgcctgtggctcccgggctggtgtttcgggagtgtccagagagcctggtctccagccgcccccgggaggagagccctgctgcccaggcgctgttgacagcggcggaaagcagcggtacccacgcgcccgccgggggaagtcggcgagcggctgcagcagcaaagaactttcccggctgggaggaccggagacaagtggcagagtcccggagccaacttttgcaagcctttcctgcgtcttaggcttctccacggcggtaaagaccagaaggcggcggagagccacgcaagagaagaaggacgtgcgctcagcttcgctcgcaccggttgttgaacttgggcgagcgcgagccgcggctgccgggcgccccctccccctagcagcggaggaggggacaagtcgtcggagtccgggcggccaagacccgccgccggccggccactgcagggtccgcactgatccgctccgcggggagagccgctgctctgggaagtgagttcgcctgcggactccgaggaaccgctgcgcacgaagagcgctcagtgagtgaccgcgacttttcaaagccgggtagcgcgcgcgagtcgacaagtaagagtgcgggaggcatcttaattaaccctgcgctccctggagcgagctggtgaggagggcgcagcggggacgacagccagcgggtgcgtgcgctcttagagaaactttccctgtcaaaggctccggggggcgcgggtgtcccccgcttgccacagccctgttgcggccccgaaacttgtgcgcgcagcccaaactaacctcacgtgaagtgacggactgttctATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCGGACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGGGAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAGCTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCGACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGCTTCGCCGAGGGCTTCGTGCGCGCCCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCAGGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGCGCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCACCTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCGAGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAGGAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCAGTTGCAAACATTTtgaagagagaccgtcgggggctgaggggcaacgaagaaaaaaaataacacagagagacagacttgagaacttgacaagttgcgacggagagaaaaaagaagtgtccgagaactaaagccaagggtatccaagttggactgggttgcgtcctgacggcgcccccagtgtgcacgagtgggaaggacttggcgcgccctcccttggcgtggagccagggagcggccgcctgcgggctgccccgctttgcggacgggctgtccccgcgcgaacggaacgttggacttttcgttaacattgaccaagaactgcatggacctaacattcgatctcattcagtattaaaggggggagggggagggggttacaaactgcaatagagactgtagattgcttctgtagtactccttaagaacacaaagcggggggagggttggggaggggcggcaggagggaggtttgtgagagcgaggctgagcctacagatgaactctttctggcctgccttcgttaactgtgtatgtacatatatatattttttaatttgatgaaagctgattactgtcaataaacagcttcatgcctttgtaagttatttcttgtttgtttgtttgggtatcctgcccagtgttgtttgtaaataagagatttggagcactctgagtttaccatttgtaataaagtatataatttttttatgttttgtttctgaaaattccagaaaggatatttaagaaaatacaataaactattggaaagtactcccctaacctcttttctgcatcatctgtagatactagctatctaggtggagttgaaagagttaagaatgtcgattaaaatcactctcagtgcttcttactattaagcagtaaaaactgttctctattagactttagaaataaatgtacctgatgtacctgatgctatggtcaggttatactcctcctcccccagctatctatatggaattgcttaccaaaggatagtgcgatgtttcaggaggctggaggaaggggggttgcagtggagagggacagcccactgagaagtcaaacatttcaaagtttggattgtatcaagtggcatgtgctgtgaccatttataatgttagtagaaattttacaataggtgcttattctcaaagcaggaattggtggcagattttacaaaagatgtatccttccaatttggaatcttctctttgacaattcctagataaaaagatggcctttgcttatgaatatttataacagcattcttgtcacaataaatgtattcaaataccaa    63          caTCR - 3 多肽1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 64          caTCR - 3 多肽2 QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 65          caTCR - 4 多肽1 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSYLNHGDYWGQGTLVTVSSQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 66          caTCR - 4 多肽2 QSVLTQPPSVSAAPGQRVTISCSGTRSNIGSDYVSWYQHLPGTAPKLLVYGDNLRPSGIPDRFSASKSGTSATLGITGLQTGDEADYYCGTWDYTLNGVVFGGGTKLTVLGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 67          caTCR - 5 多肽1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 68          caTCR - 5 多肽2 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 69          caTCR - 6 多肽1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARWHGGPYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL 70          caTCR - 6 多肽2 QPVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS 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

圖式說明具有本發明之特徵及優點的某些實施例。此等實施例並不意欲以任何方式限制所附申請專利範圍之範疇。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.

圖 1A為例示性caTCR及CSR對(上圖)及用於本文所描述之T細胞之活體內功效研究的兩種表現構築體(下圖)的示意性圖示。構築體1編碼抗GPC3 caTCR (「抗GPC3-caTCR」)加包含源於CD30之協同刺激域的抗GPC3 CSR (「抗GPC3-CD30-CSR」)，而構築體2編碼「抗GPC3-caTCR」+「抗GPC3-CD30-CSR」及「c-Jun」多肽。 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.

圖 1B為兩種不同小鼠模型中的表現抗GPC3-caTCR及抗GPC3-CD30-CSR之T細胞在存在或不存在c-Jun過度表現之情況下的活體內功效研究設計之示意性圖示。 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.

圖 2為展示HepG2-luc小鼠模型中的抗GPC3-caTCR加抗GPC3-CD30-CSR T細胞在存在或不存在c-Jun過度表現之情況下的活體內腫瘤殺傷效果之圖式。自兩個健康人類供體分離T細胞。對於各T細胞類型，測試兩個劑量(5×10 6及2×10 6)。 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.

圖 3為展示在腫瘤再攻擊研究中HepG2-luc小鼠模型中的抗GPC3-caTCR加抗GPC3-CD30-CSR T細胞在存在或不存在c-Jun過度表現之情況下的活體內腫瘤殺傷效果之圖式，其中在初始腫瘤消退之後，給動物再次植入腫瘤細胞。 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.

圖 4A 至圖 4C為展示注射有抗GPC3-caTCR加抗GPC3-CD30-CSR T細胞之HepG2-luc及Hep3B小鼠在存在或不存在c-Jun過度表現之情況下的周邊血液T細胞計數之圖式。在 圖 4A中，自供體A分離T細胞且小鼠模型為HepG2-luc；在 圖 4B中，自供體B分離T細胞且小鼠模型為HepG2-luc；在 圖 4C中，自供體C分離T細胞且小鼠模型為Hep3B。 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.

圖 5為展示Hep3B小鼠模型中的抗GPC3-caTCR加抗GPC3-CD30-CSR T細胞在存在或不存在c-Jun過度表現之情況下的活體內腫瘤殺傷效果之圖式。 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.xmlTW202413646A_112126959_SEQL.xml

Claims (83)

一或多種表現構築體，其包含一或多個用於表現以下之表現卡匣： a)嵌合抗體T細胞受體(TCR)構築體(caTCR)，其包含： i)特異性結合磷脂醯肌醇蛋白聚糖3 (glypican3, GPC3)的抗原結合模組；及 ii) TCR模組(TCRM)，其包含有包含第一TCR跨膜域(TCR-TM)之第一TCR域(TCRD)及包含第二TCR-TM之第二TCRD，其中該TCRM促進至少一種TCR相關信號傳導分子的募集； b)嵌合刺激受體(CSR)，其包含： i)能夠與GPC3結合或相互作用的配位體結合模組； ii)跨膜模組；及 iii)能夠向該免疫細胞提供協同刺激信號之協同刺激免疫細胞信號傳導模組，其中該配位體結合模組及該協同刺激免疫細胞信號傳導模組並不源於相同分子，且其中該CSR缺乏功能性初級免疫細胞信號傳導域；及 c)人類c-Jun多肽。 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. 如請求項1之表現構築體，其中該c-Jun為野生型人類c-Jun。The expression construct of claim 1, wherein the c-Jun is wild-type human c-Jun. 如請求項2之表現構築體，其中該野生型人類c-Jun與SEQ ID NO: 1之胺基酸序列具有至少約90%一致性。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. 如請求項1之表現構築體，其中該c-Jun為突變型人類c-Jun。The expression construct of claim 1, wherein the c-Jun is a mutant human c-Jun. 如請求項4之表現構築體，其中該突變型人類c-Jun在其反式活化域或δ域中包含不活化突變。The expression construct of claim 4, wherein the mutant human c-Jun comprises an inactivating mutation in its transactivation domain or delta domain. 如請求項4或5之表現構築體，其中該突變型人類c-Jun包含： (i)相比於SEQ ID NO: 1之S63A及S73A取代；或 (ii)相比於SEQ ID NO: 1之胺基酸殘基2與102之間或胺基酸殘基30與50之間的缺失。 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. 如請求項1至6中任一項之表現構築體，其中該GPC3為細胞表面結合之GPC3。The expression construct of any one of claims 1 to 6, wherein the GPC3 is cell surface-bound GPC3. 如請求項1至7中任一項之表現構築體，其中該TCRM源於人類γ/δ TCR。The expression construct of any one of claims 1 to 7, wherein the TCRM is derived from a human γ/δ TCR. 如請求項1至8中任一項之表現構築體，其中該caTCR進一步包含穩定模組，該穩定模組包含第一穩定域及第二穩定域，其中該第一穩定域及該第二穩定域彼此具有使該caTCR穩定的結合親和力。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. 如請求項9之表現構築體，其中該穩定模組係選自由以下組成之群：TCR恆定域、C H1-C L模組、C H2-C H2模組、C H3-C H3模組及C H4-C H4模組。 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. 如請求項10之表現構築體，其中該穩定模組係衍生自人類蛋白質。The expression construct of claim 10, wherein the stabilization module is derived from a human protein. 如請求項10或11之表現構築體，其中該C H1-C L模組中所含之C L係衍生自κ抗體輕鏈或λ抗體輕鏈。 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. 如請求項1至12中任一項之表現構築體，其中該caTCR之抗原結合模組為Fab、Fab'、(Fab') 2、Fv或單鏈Fv (scFv)。 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). 如請求項1至13中任一項之表現構築體，其中該caTCR之抗原結合模組包含： (i)包含有包含SEQ ID NO: 6之胺基酸序列的重鏈互補決定區(HC-CDR) 1 (HC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的重鏈可變域(V H)；及包含有包含SEQ ID NO: 9之胺基酸序列的輕鏈互補決定區(LC-CDR) 1 (LC-CDR1)或其包含至多約3個胺基酸取代之變異體、包含GDN之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的輕鏈可變域(V L)； (ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含YDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L；或 (iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含DDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L。 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. 如請求項14之表現構築體，其中該caTCR之抗原結合模組包含： (i)包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L； (ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；或 (iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。 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. 如請求項1至15中任一項之表現構築體，其中該caTCR之抗原結合模組包含： (i)包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25具有至少約90%序列一致性之胺基酸序列的V L； (ii)包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27具有至少約90%序列一致性之胺基酸序列的V L；或 (iii)包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29具有至少約90%序列一致性之胺基酸序列的V L。 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. 如請求項16之表現構築體，其中caTCR之抗原結合模組包含： (i)包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L； (ii)包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；或 (iii)包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 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. 如請求項1至17中任一項之表現構築體，其中該caTCR為雜二聚體，其包含有包含第一TCRD之第一多肽鏈及包含第二TCRD之第二多肽鏈。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. 如請求項1至18中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V L。 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. 如請求項1至18中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含與衍生自TCR γ鏈之TCRD融合的V H，該第二多肽鏈包含與衍生自TCR δ鏈之TCRD融合的V L。 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. 如請求項1至19中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 30之胺基酸序列或與SEQ ID NO: 30具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 31之胺基酸序列或與SEQ ID NO: 31具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至18及20中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 10之胺基酸序列或與SEQ ID NO: 10具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 5之胺基酸序列或與SEQ ID NO: 5具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至19中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 63之胺基酸序列或與SEQ ID NO: 63具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 64之胺基酸序列或與SEQ ID NO: 64具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至18及20中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 65之胺基酸序列或與SEQ ID NO: 65具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 66之胺基酸序列或與SEQ ID NO: 66具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至19中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 67之胺基酸序列或與SEQ ID NO: 67具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 68之胺基酸序列或與SEQ ID NO: 68具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至18及20中任一項之表現構築體，其中該caTCR為包含第一多肽鏈及第二多肽鏈的雜二聚體，該第一多肽鏈包含SEQ ID NO: 69之胺基酸序列或與SEQ ID NO: 69具有至少約90%序列一致性之胺基酸序列，該第二多肽鏈包含SEQ ID NO: 70之胺基酸序列或與SEQ ID NO: 70具有至少約90%序列一致性之胺基酸序列。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. 如請求項1至26中任一項之表現構築體，其中該CSR之跨膜模組包含衍生自CD30、CD28、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137或CD154之跨膜域的跨膜域。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. 如請求項1至27中任一項之表現構築體，其中該CSR之跨膜模組包含衍生自CD30之跨膜域的跨膜域。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. 如請求項1至28中任一項之表現構築體，其中該協同刺激免疫細胞信號傳導模組衍生自TCR之協同刺激受體的胞內域。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. 如請求項29之表現構築體，其中該協同刺激受體係選自由CD30、CD28、4-IBB、OX40、ICOS、CD27及CD40組成之群。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. 如請求項1至30中任一項之表現構築體，其中該CSR之協同刺激免疫細胞信號傳導模組衍生自人類CD30。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. 如請求項31之表現構築體，其中人類CD30之協同刺激免疫細胞信號傳導模組包含SEQ ID NO: 44之胺基酸序列。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. 如請求項1至32中任一項之表現構築體，其中該CSR之協同刺激免疫細胞信號傳導模組及跨膜域均衍生自人類CD30。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. 如請求項1至33中任一項之表現構築體，其中該CSR之配位體結合模組包含： (i)包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含GDN之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L； (ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含YDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L；或 (iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1或其包含至多約3個胺基酸取代之變異體、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3或其包含至多約3個胺基酸取代之變異體的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1或其包含至多約3個胺基酸取代之變異體、包含DDS之胺基酸序列的LC-CDR2或其包含至多約3個胺基酸取代之變異體及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3或其包含至多約3個胺基酸取代之變異體的V L。 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. 如請求項34之表現構築體，其中該CSR之配位體結合模組包含： (i)包含有包含SEQ ID NO: 6之胺基酸序列的HC-CDR1、包含SEQ ID NO: 7之胺基酸序列的HC-CDR2及包含SEQ ID NO: 8之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 9之胺基酸序列的LC-CDR1、包含GDN之胺基酸序列的LC-CDR2及包含SEQ ID NO: 11之胺基酸序列的LC-CDR3的V L； (ii)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L；或 (iii)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L。 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. 如請求項1至35中任一項之表現構築體，其中該CSR之配位體結合模組包含： (i)包含SEQ ID NO: 24之胺基酸序列或與SEQ ID NO: 24具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 25之胺基酸序列或與SEQ ID NO: 25具有至少約90%序列一致性之胺基酸序列的V L； (ii)包含SEQ ID NO: 26之胺基酸序列或與SEQ ID NO: 26具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 27之胺基酸序列或與SEQ ID NO: 27具有至少約90%序列一致性之胺基酸序列的V L；或 (iii)包含SEQ ID NO: 28之胺基酸序列或與SEQ ID NO: 28具有至少約90%序列一致性之胺基酸序列的V H，及包含SEQ ID NO: 29之胺基酸序列或與SEQ ID NO: 29具有至少約90%序列一致性之胺基酸序列的V L。 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. 如請求項36之表現構築體，其中該CSR之配位體結合模組包含： (i)包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L； (ii)包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；或 (iii)包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 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. 如請求項1至37中任一項之表現構築體，其中該CSR之配位體結合模組包含： (i) SEQ ID NO: 32或與SEQ ID NO: 32具有至少約90%序列一致性之胺基酸序列； (ii) SEQ ID NO: 33或與SEQ ID NO: 33具有至少約90%序列一致性之胺基酸序列；或 (iii) SEQ ID NO: 34或與SEQ ID NO: 34具有至少約90%序列一致性之胺基酸序列。 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. 如請求項1至38中任一項之表現構築體，其中： (i)該caTCR之抗原結合模組包含： (a)包含有包含SEQ ID NO: 18之胺基酸序列的HC-CDR1、包含SEQ ID NO: 19之胺基酸序列的HC-CDR2及包含SEQ ID NO: 20之胺基酸序列的HC-CDR3的V H；及 (b)包含有包含SEQ ID NO: 21之胺基酸序列的LC-CDR1、包含DDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 23之胺基酸序列的LC-CDR3的V L；及 (ii)該CSR之配位體結合模組包含： (a)包含有包含SEQ ID NO: 12之胺基酸序列的HC-CDR1、包含SEQ ID NO: 13之胺基酸序列的HC-CDR2及包含SEQ ID NO: 14之胺基酸序列的HC-CDR3的V H；及 (b)包含有包含SEQ ID NO: 15之胺基酸序列的LC-CDR1、包含YDS之胺基酸序列的LC-CDR2及包含SEQ ID NO: 17之胺基酸序列的LC-CDR3的V L。 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 . 如請求項1至39中任一項之表現構築體，其中該caTCR之抗原結合模組包含結合GPC3之Fab，其中該CSR之配位體結合模組包含結合GPC3之scFv，且其中該CSR之跨膜模組及協同刺激免疫細胞信號傳導模組均衍生自CD30。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. 如請求項40之表現構築體，其中該CSR包含CD30之片段，該片段包含SEQ ID NO: 43之胺基酸序列，且其中： (1)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L； (2)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L； (3)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L； (4)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L； (5)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L； (6)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L； (7)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L； (8)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；或 (9)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L。 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. 如請求項40或41之表現構築體，其中該CSR包含CD30之片段，該片段包含SEQ ID NO: 43之胺基酸序列，且其中： (1)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 33之胺基酸序列； (2)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 32之胺基酸序列； (3)    (i)該Fab包含有包含SEQ ID NO: 28之胺基酸序列的V H及包含SEQ ID NO: 29之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 34之胺基酸序列； (4)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 32之胺基酸序列； (5)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 33之胺基酸序列； (6)    (i)該Fab包含有包含SEQ ID NO: 26之胺基酸序列的V H及包含SEQ ID NO: 27之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 34之胺基酸序列； (7)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 32之胺基酸序列； (8)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 33之胺基酸序列；或 (9)    (i)該Fab包含有包含SEQ ID NO: 24之胺基酸序列的V H及包含SEQ ID NO: 25之胺基酸序列的V L；及 (ii)該scFv包含SEQ ID NO: 34之胺基酸序列。 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. 如請求項1至42中任一項之表現構築體，其中該(等)構築體為病毒載體。The expression construct of any one of claims 1 to 42, wherein the construct(s) is a viral vector. 如請求項43之表現構築體，其中該(等)病毒載體係選自由以下組成之群：慢病毒載體、腺病毒載體、腺相關病毒載體、牛痘載體、單純疱疹病毒載體及EB病毒載體(Epstein-Barr 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. 如請求項1至42中任一項之表現構築體，其中該表現構築體包含用於表現該caTCR、該CSR及該c-Jun之多順反子表現卡匣。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. 一種多順反子表現構築體，其包含用於表現以下之表現卡匣： a)嵌合抗體T細胞受體(TCR)構築體(caTCR)，其包含： i)特異性結合磷脂醯肌醇蛋白聚糖3 (GPC3)的抗原結合模組；及 ii)衍生自人類γ/δ TCR之TCR模組(TCRM)； b)嵌合刺激受體(CSR)，其包含： i)能夠與GPC3結合或相互作用的配位體結合模組； ii)跨膜模組；及 iii)衍生自人類CD30之胞內域的協同刺激免疫細胞信號傳導模組；及 c)人類c-Jun多肽。 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. 如請求項46之表現構築體，其中該表現卡匣包含SEQ ID NO: 1之編碼序列及： (i) SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 33之編碼序列； (ii) SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 32之編碼序列； (iii) SEQ ID NO: 28及29之編碼序列及SEQ ID NO: 34之編碼序列； (iv) SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 32之編碼序列； (v) SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 33之編碼序列； (vi) SEQ ID NO: 26及27之編碼序列及SEQ ID NO: 34之編碼序列； (vii) SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 32之編碼序列； (viii) SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 33之編碼序列；或 (ix) SEQ ID NO: 24及25之編碼序列及SEQ ID NO: 34之編碼序列。 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. 如請求項47之表現構築體，其中編碼序列在框內藉由2A編碼序列或藉由內部核糖體進入位點(IRES)分開。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). 如請求項46至48中任一項之表現構築體，其中該表現卡匣包含組成性或可誘導型啟動子。A performance construct as in any one of claims 46 to 48, wherein the performance cartridge comprises a constitutive or inducible promoter. 如請求項49之表現構築體，其中該啟動子為EF-1α啟動子。The expression construct of claim 49, wherein the promoter is the EF-1α promoter. 如請求項46至50中任一項之表現構築體，其中該構築體為病毒載體。The expression construct of any one of claims 46 to 50, wherein the construct is a viral vector. 如請求項51之表現構築體，其中該病毒載體係選自由以下組成之群：慢病毒載體、腺病毒載體、腺相關病毒載體、牛痘載體、單純疱疹病毒載體及EB病毒載體。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. 一種重組病毒，其包含如請求項50至52中任一項之多順反子表現構築體。A recombinant virus comprising the multicistronic expression construct of any one of claims 50 to 52. 如請求項53之重組病毒，其中該多順反子表現構築體為慢病毒載體。The recombinant virus of claim 53, wherein the polycistronic expression construct is a lentiviral vector. 一種工程改造免疫細胞之方法，其包含： (a)提供起始細胞群， (b)將如請求項1至52中任一項之表現構築體或如請求項53或54之重組病毒引入該起始細胞群中， (c)視情況選擇表現該caTCR、該CSR及該c-Jun之細胞，及 (d)自該等步驟(b)或(c)之細胞獲得經工程改造之免疫細胞。 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). 如請求項55之方法，其中該免疫細胞為T細胞。The method of claim 55, wherein the immune cell is a T cell. 如請求項56之方法，其中該T細胞係選自由以下組成之群：細胞毒性T細胞、輔助性T細胞、自然殺手T細胞及抑制性T細胞。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. 如請求項55至57中任一項之方法，其中該起始細胞群包含免疫細胞。The method of any one of claims 55 to 57, wherein the starting cell population comprises immune cells. 如請求項55至58中任一項之方法，其中該起始細胞群包含自體或同種異體T細胞。The method of any one of claims 55 to 58, wherein the starting cell population comprises autologous or allogeneic T cells. 如請求項55至57中任一項之方法，其中該起始細胞群包含多能或多潛能細胞，且步驟(d)包含使該等步驟(b)或(c)之細胞分化成免疫細胞。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. 一種人類細胞群，其包含如請求項1至52中任一項之表現構築體或如請求項53或54之重組病毒。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. 如請求項61之人類細胞群，其中該等人類細胞為免疫細胞。The human cell population of claim 61, wherein the human cells are immune cells. 一種免疫細胞群，其係藉由如請求項55至60中任一項之方法獲得。An immune cell population obtained by the method of any one of claims 55 to 60. 如請求項63之免疫細胞群，其中該等免疫細胞為人類細胞。The immune cell population of claim 63, wherein the immune cells are human cells. 如請求項62至64中任一項之免疫細胞，其中該等免疫細胞為T細胞。The immune cell of any one of claims 62 to 64, wherein the immune cells are T cells. 如請求項65之免疫細胞，其中該等T細胞為CD8 +T細胞。 The immune cell of claim 65, wherein the T cells are CD8 + T cells. 如請求項62至66中任一項之免疫細胞，其中該等免疫細胞與不過度表現c-Jun之對應細胞相比表現較少量之耗竭標記物。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. 如請求項67之免疫細胞，其中該耗竭標記物係選自由CD39、PD-1、TIM-3及LAG-3組成之群。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. 一種醫藥組合物，其包含如請求項1至52中任一項之表現構築體、如請求項53或54之重組病毒或如請求項61至68中任一項之細胞及醫藥學上可接受之載劑。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. 一種殺傷目標細胞之方法，其包含使該等目標細胞與如請求項69之醫藥組合物在允許由免疫細胞殺傷該等目標細胞之條件下接觸，其中該等目標細胞表現GPC3。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. 如請求項70之方法，其中該等免疫細胞與不包含引起c-Jun過度表現之外源性核酸分子的對應免疫細胞相比，在與該等目標細胞接觸時表現較少量之耗竭標記物。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. 如請求項70或71之方法，其中該等免疫細胞為T細胞。The method of claim 70 or 71, wherein the immune cells are T cells. 如請求項72之方法，其中該等T細胞為CD8 +T細胞。 The method of claim 72, wherein the T cells are CD8 + T cells. 如請求項73之方法，其中該等目標細胞為癌細胞。The method of claim 73, wherein the target cells are cancer cells. 如請求項74之方法，其中該CSR中之協同刺激免疫細胞信號傳導模組係衍生自人類CD30，且該等免疫細胞與經工程改造以表現其協同刺激免疫細胞信號傳導模組係衍生自人類CD28的CSR之對應免疫細胞相比，表現較少量之耗竭標記物。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. 如請求項70至75中任一項之方法，其中該耗竭標記物係選自由CD39、PD-1、TIM-3及LAG-3組成之群。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. 一種治療有需要之個體之方法，其包含向該個體投與如請求項69之醫藥組合物。A method for treating a subject in need thereof, comprising administering the pharmaceutical composition of claim 69 to the subject. 一種如請求項1至52中任一項之表現構築體、如請求項53或54之重組病毒或如請求項69之醫藥組合物的用途，其用於製造供治療有需要之個體用之藥劑。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. 一種如請求項1至52中任一項之表現構築體、如請求項53或54之重組病毒或如請求項69之醫藥組合物的用途，其用於治療有需要之個體。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. 如請求項77之方法或如請求項78或79之用途，其中該個體患有GPC3陽性疾病。The method of claim 77 or the use of claim 78 or 79, wherein the individual suffers from a GPC3-positive disease. 如請求項80之方法或用途，其中該GPC3陽性疾病為癌症。The method or use of claim 80, wherein the GPC3-positive disease is cancer. 如請求項81之方法或用途，其中該癌症係選自由以下組成之群：肝細胞癌(HCC)、黑色素瘤、肺癌、非小細胞肺癌、胰臟癌、乳癌、三陰性乳癌、肺鱗狀細胞癌、卵巢癌、卵黃囊瘤、絨膜癌、神經母細胞瘤、肝母細胞瘤、威爾姆斯氏瘤(Wilms' tumor)、睪丸非精原細胞生殖細胞瘤、胃癌及脂肪肉瘤。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. 一種減少經工程改造之免疫細胞耗竭的方法，其包含向該經工程改造之免疫細胞中引入增加該細胞中c-Jun之表現的外源性核酸分子，其中該經工程改造之免疫細胞包含如請求項1至52中任一項之表現構築體。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.
TW112126959A 2022-07-22 2023-07-19 Immune cell therapy TW202413646A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US63/369,171 2022-07-22

Publications (1)

Publication Number Publication Date
TW202413646A true TW202413646A (en) 2024-04-01

Family

ID=

Similar Documents

Publication Publication Date Title
JP7451627B2 (en) Chimeric receptor and its use
JP7157839B2 (en) Combination of immunotherapy and cytokine control therapy for cancer treatment
US11718685B2 (en) Compositions and methods for targeting stromal cells for the treatment of cancer
US10780120B2 (en) Prostate-specific membrane antigen cars and methods of use thereof
EP3842450A1 (en) Antibody/t-cell receptor chimeric constructs and uses thereof
JP6985934B2 (en) Manipulated hematopoietic stem / progenitor cells and non-T effector cells, as well as their use
US11780899B2 (en) Engineered proteins to enhance sensitivity of a cell to IL-2
JP2022514023A (en) Methods and Uses for Expanding Tumor-Infiltrating Lymphocytes Using Manipulated Cytokine Receptor Pairs
US20220133795A1 (en) Expansion of Tumor Infiltrating Lymphocytes From Liquid Tumors and Therapeutic Uses Thereof
TW202305118A (en) Cish gene editing of tumor infiltrating lymphocytes and uses of same in immunotherapy
CN116745313A (en) Reprogramming immune cells by targeted integration of zeta-deficient chimeric antigen receptor transgenes
US20210060070A1 (en) Adoptive cell therapy and methods of dosing thereof
US20230158072A1 (en) Combination therapy involving anti-cd39 antibodies and adoptive cell therapy
TW202239768A (en) Improved immune cell therapy
JP2024509917A (en) Method for selective stimulation of T cells in solid tumors using orthogonal IL-2 delivery by oncolytic viruses
TW202413646A (en) Immune cell therapy
WO2024020429A1 (en) Immune cell therapy
KR20220095228A (en) Methods of Producing Cytotoxic Effector Memory T Cells for the Treatment of T Cells in Cancer