Chimeric antigen receptor (CAR) of TCR beta chain variable region

Abstract

The present invention relates to chimeric antigen receptors (CAR) themselves, CAR constructs and T cell receptor (TCR) beta chain variable region beta (TCRV beta) CAR. The present invention relates to the use of CARs in immunotherapy, for example in the treatment, prevention or amelioration of cancer, for example T cell malignancies, as well as diseases caused or involved by pathogenic T cells, for example autoimmune diseases. In particular, the present invention relates to conventional T cells and constant natural killer T (iNKT) cells expressing anti-TCRV [beta] CAR and methods for their preparation. The present invention relates to nucleic acids and vectors encoding anti-TCRV [beta] CAR, pharmaceutical compositions comprising the constructs and cells, and medical uses of the compositions, anti-TCRV [beta] CAR constructs, and T cells and iNKT cells expressing anti-TCRV [beta] CAR.

Description

Technical Field

[0001] The present invention relates to chimeric antigen receptors (CARs) themselves and CAR constructs, in particular (but not limited to) T cell receptor (TCR) β chain variable region β (TCRVβ) CAR. The present invention relates to the use of CAR in immunotherapy, such as treating, preventing or improving cancer (e.g., T cell malignancies) and diseases caused or involved by pathogenic T cells (e.g., autoimmune diseases). The present invention particularly relates to conventional T cells and constant natural killer T (iNKT) cells expressing anti-TCRVβCAR and methods for preparing them. The present invention relates to nucleic acids and vectors encoding anti-TCRVβCAR, pharmaceutical compositions comprising constructs and cells, and medical uses of compositions, anti-TCRVβCAR constructs, and T cells and iNKT cells expressing anti-TCRVβCAR. Background Art

[0002] Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia / lymphoma (ATL) and most other T-cell lymphomas (TCL) have a poor prognosis with current treatments. Carriers of HTLV-1, a virus prevalent in many parts of the world, have a 5% lifetime risk of developing ATL. 2,3 .

[0003] Cell surface T cell receptor (TCR) α / β chain heterodimers are restricted to expression on normal T cells, non-malignant pathogenic T cells, and malignant T cells. The TCR variable region β (TCRVβ) subunit is encoded by the TRBV gene. Humans have 23 families and 114 alleles of the TRBV gene, each of which contains 0.5-9% of the normal T cell pool, of which TCRVβ2 is the most common. Signals from the TCR determine the cell fate of normal T cells. Components of the TCR signaling pathway are frequently mutated in TCL (including ATL), indicating that the TCR plays a driving role in TCL carcinogenesis. Surface TCR in blood and lymph node lymphoma T cells 4–6 and stable expression on non-malignant pathogenic T cells.

[0004] CAR-T therapy for B-cell leukemias and lymphomas targeting pan-B cell markers (e.g., CD19 and CD20) can achieve sustained clinical remissions in up to 40% of patients with relapsed / refractory disease, but at the expense of clinically tolerable pan-B cell depletion and hypogammaglobulinemia 7. However, pan-T cell depletion results in severe immunosuppression that is clinically intolerable. Since most TCLs are CD4+, anti-CD4 CAR-T therapy has also been proposed for these lymphomas (e.g., see clinical trial NCT 03829540). This approach also results in immunosuppression similar to acquired immunodeficiency syndrome unless rescued by stem cell transplantation. Other CAR targets include CD5 and CD7; however, these targets are widely expressed on healthy T cells and may lead to fratricide reactions unless the CAR effector cells are additionally genetically edited to lack expression of these markers. 8 Recently, the effectiveness of using CAR-T cells to target the TCRβ chain constant region 1, which is expressed in approximately 40–60% of TCRαβ lymphocytes, has been reported. 9 The impact of depleting 40-60% of the T cell repertoire on the immune status of an individual remains to be seen. It is possible that the loss of half of the TCR repertoire, as reported in mouse models, would severely impair immunity in humans. 10 Current anti-T-cell lymphoma CAR-based immunotherapies eliminate all or 50% of healthy T cells, rendering patients with T-cell lymphoma severely or profoundly immunosuppressed.

[0005] Therefore, there is a need to provide improved immunotherapies for T cell malignancies, such as T cell lymphomas (including ATL), improved immunotherapies for treating diseases caused directly or indirectly by pathogenic non-malignant T cells, such as certain autoimmune diseases, and improved immunotherapies for targeting normal, non-pathogenic T cells. Summary of the invention

[0006] To combat malignant and non-malignant pathogenic T cells as well as non-pathogenic T cells, the inventors developed CAR-iNKT (invariant natural killer T) cells as an "off-the-shelf" allogeneic immunotherapy. iNKT cells are a rare (i.e., they represent less than 0.1% of the total T cells) and evolutionarily conserved T cell subset that possesses features of both innate and adaptive immune responses. 11–13 In humans, iNKTs are characterized by the expression of a constant TCR Vα24Jα18 chain, which is predominantly paired with a diverse TCR Vβ11 chain (iTCR). 14 iNKT cells are restricted by CD1d, a non-polymorphic glycolipid-presenting HLA class I-like molecule expressed on monocytes, macrophages, dendritic cells, B cells, thymocytes, and some epithelial tissues. 15 In addition, iNKT cells have a memory effector phenotype and can migrate to extralymphatic tissues. 18–20, where it regulates multiple immune responses, including anti-tumor and anti-pathogen responses 21,22 .

[0007] Because iNKT cells can prevent acute graft-versus-host disease (aGVHD) 25–28 , CAR-iNKT cell immunotherapy can be derived from allogeneic healthy donors as an "off-the-shelf" treatment without the need to delete endogenous TCRs like conventional T cells. In contrast, standard autologous CAR-T immunotherapy may be limited by financial and logistical challenges and the poor fitness of patient-derived T cells.

[0008] Based on the above considerations, the inventors explored targeting the ATL / TCL-specific TCRVβ chain by CAR engineering of T cells and iNKT cells. In order to meet the urgent clinical needs for new treatment methods, the inventors developed an exemplary chimeric antigen receptor (CAR) that targets a group of subunits of the T cell receptor (TCR) β chain that are constitutively expressed by mature T cell leukemias and lymphomas.

[0009] Thus, a first aspect of the present invention provides a chimeric antigen receptor (CAR) construct specific for the variable region (Vβ) subunit of the β chain of a T cell receptor (TCR) of a T cell.

[0010] As shown in the examples, the inventors have developed various embodiments of exemplary CARs specific for various TCR Vβ subunits of the present invention (herein, for TCR Vβ1, referred to as clone BL37.2; for TCR Vβ2, referred to as clone MPB2D5; for TCR Vβ9, referred to as clone FIN9; for TCR Vβ11, referred to as clone C21). Advantageously, using conventional T cells and innate natural killer (iNKT) cells as CAR effector cells, the inventors unexpectedly demonstrated that anti-VβCAR-T cells and CAR-iNKT cells carrying the CAR of the present invention can successfully kill in vitro (in vitro) expanded primary T cells and ex vivo (ex vivo) adult T cell leukemia (ATL) cells expressing the corresponding TCRVβ subunit, while unexpectedly minimizing off-target killing of healthy T cells expressing other TCRVβ subunits. The inventors have successfully demonstrated that CAR-iNKT activity is significantly enhanced when challenged with a target expressing CD1d that has been pulsed with α-galactosylceramide, a selective ligand that activates iNKT cells. Ligation of anti-VβCAR induced degranulation of T cells and iNKT effector cells, secretion of IFN-γ and TNF-α, and upregulation of perforin and granzyme expression. Targeting T cells expressing a single Vβ subunit avoids virus-specific CTLs. In addition, in a brief in vitro assay, the inventors have demonstrated that CAR-mediated killing has no effect on the expression of the virus that causes ATL (human T-cell leukemia virus type 1). Finally, in a subcutaneous model of T-cell lymphoma, anti-TCRVβCAR-iNKT (but not anti-CD19CAR-iNKT) significantly reduced tumor size (p<0.01). Tumor-bearing mice that received iNKT effector cells showed no signs of graft-versus-host disease.

[0011] Therefore, through these experiments, the inventors unexpectedly demonstrated that CAR targeting the TCRVβ subunit can effectively kill malignant T cell clones while minimizing off-target cytotoxicity. In the presence of iNKT ligands, CAR-iNKT effector cells exhibit enhanced killing activity, reduce the risk of acute graft-versus-host disease, and are suitable as a ready-made product for infusion into third-party donors.

[0012] In one embodiment, the CAR construct is specific for the T cell receptor (TCR) β chain variable region (Vβ) subunit of normal, non-pathogenic T cells.

[0013] However, in another embodiment, the CAR construct is specific for the T cell receptor (TCR) β chain variable region (Vβ) subunit of pathogenic T cells.

[0014] Pathogenic T cells are clonal in nature to tumor cells, sharing expression of a single TCRVβ subunit, with no bias or preference in the use of the TCRVβ subunit family 4–6 . Advantageously, clonal targeting of specific TCRVβ subunits expressed by pathogenic T cells provides a highly selective and tumor-specific therapeutic solution, with “on-target off-tumour” toxicity limited to less than 5-9% of normal T cells. Since the expression of each TCRVβ subunit is restricted to less than 9% of healthy T cells, this approach avoids treatment-induced immunosuppression and immune dysregulation that may be caused by alternative CAR-based immunotherapies for ATL / TCL, as the new construct retains more than 90% of healthy T cells.

[0015] Therefore, based on the above considerations, the inventors explored targeting the ATL / TCL-specific TCRVβ chain through CAR engineering of T cells and iNKT cells. In order to meet the urgent clinical needs for new treatment methods, the inventors developed a chimeric antigen receptor (CAR) targeting the T cell receptor (TCR) β chain, which is constitutively expressed by mature T cell leukemias and lymphomas.

[0016] Therefore, preferably, the CAR construct of the present invention is specific for the TCRVβ subunit of pathogenic T cells.

[0017] "Pathogenic T cells" can refer to T cells expressed in clinical diseases, such as T cell malignancies (including TCL and ATL), infections, and autoimmune diseases. Therefore, anti-TCRVβCAR can target pathogenic T cells, such as T cells involved in T cell lymphomas and T cell leukemias, and T cells that cause autoimmune diseases in a highly selective manner.

[0018] Thus, the pathogenic T cell may be a malignant pathogenic T cell. Alternatively, the pathogenic T cell may be a non-malignant pathogenic T cell.

[0019] Healthy T cells express a family of 23 different TCRVβ-chain molecules.

[0020] Advantageously, each T cell expresses only one TCRVβ chain. Therefore, pathological T cells (e.g., T cells in cancer, i.e., T cell lymphomas and T cell leukemias, and T cells causing autoimmune diseases) express only one type of TCRVβ chain. However, the same TCRVβ chain expressed in pathological T cells is also expressed in less than 10% of normal healthy T cells. Therefore, although more than 90% of the patient's healthy T cells and T cell-dependent immunity will remain intact, the anti-TCRVβCAR that selectively targets pathological T cells will also target less than 10% of the patient's healthy T cells. Therefore, in some embodiments, the CAR construct of the present invention can target healthy T cells.

[0021] Table 1 below lists the TCRVβ subunits on T cells and their associated encoding genes, any one or more of which can be targeted by the CAR construct of the present invention.

[0022] Table 1 - Variable subunits of β chains on T cells (Vβ)

[0023]

[0024]

[0025]

[0026]

[0027] Therefore, preferably, the Vβ subunit can be selected from the Vβ subunits shown in Table 1.

[0028] As shown in Table 1, the frequency of use of the TCRVβ chain family in T cell lymphoma is similar to that in the normal T cell library. In a preferred embodiment, the CAR construct targets the TCRVβ subunit on the T cell, wherein the subunit is any one of the left column of Table 1.

[0029] As described in the examples, the inventors prepared four embodiments of CAR constructs, each of which is specific to TCR-Vβ1, TCR-Vβ2, TCR-Vβ9 and TCR-Vβ11. Therefore, preferably, the CAR construct targets the TCRVβ subunit on the T cell, which is selected from the following Vβ subunits: TCR-Vβ1, TCR-Vβ2, TCR-Vβ9 and TCR-Vβ11.

[0030] Therefore, preferably, the CAR construct is specific for TCR-Vβ1. One embodiment of the polypeptide sequence of the TCR Vβ1 subunit (encoded by H. sapiens TCRBV9, of which there are three alleles, TRBV9*01 to *039-UniProtKB:A0A0B4J1U6) is represented herein as SEQ ID No: 1 as shown below:

[0031] GVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYNGEERAK GNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSV

[0032] [SEQ ID No:1]

[0033] Therefore, preferably, the CAR construct is specific for a TCR-Vβ1 subunit comprising an amino acid sequence substantially as shown in SEQ ID No: 1 or a variant or fragment thereof.

[0034] Preferably, the CAR construct is specific to TCR-Vβ2. An example scheme of the polypeptide sequence of the TCR Vβ2 subunit (encoded by Homo sapiens TCRBV20-1, of which there are seven alleles, TRBV20-1*01 to *07-UniProtKB:A0A075B6N2) is represented herein as SEQ ID No: 2 as shown below:

[0035] AVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKAT YEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAR

[0036] [SEQ ID No:2]

[0037] Therefore, preferably, the CAR construct is specific for a TCR-Vβ2 subunit comprising an amino acid sequence substantially as shown in SEQ ID No: 2 or a variant or fragment thereof.

[0038] Preferably, the CAR construct is specific for TCR-Vβ9. An embodiment of the polypeptide sequence of the TCR Vβ9 subunit (encoded by Homo sapiens TCRBV3-1, of which there are two alleles, TRBV3-1*01 and *02-UniProtKB / Swiss-Prot: A0A576) is represented herein as SEQ ID No: 3 as shown below:

[0039] AVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELIIN ETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSQ

[0040] [SEQ ID No:3]

[0041] Therefore, preferably, the CAR construct is specific for a TCR-Vβ9 subunit comprising an amino acid sequence substantially as shown in SEQ ID No: 3 or a variant or fragment thereof.

[0042] Preferably, the CAR construct is specific for TCR-Vβ11. One embodiment of the polypeptide sequence of the TCR Vβ11 subunit (encoded by Homo sapiens TCRBV25-1*01, which has one allele - UniProtKB: A0A075B6N4) is represented herein as SEQ ID No: 4 as shown below:

[0043] DIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEK GDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSE

[0044] [SEQ ID No:4]

[0045] Therefore, preferably, the CAR construct is specific for the TCR-Vβ11 subunit, which TCR-Vβ11 subunit comprises an amino acid sequence substantially as shown in SEQ ID No: 4 or a variant or fragment thereof.

[0046] Preferably, in one embodiment, the CAR construct comprises:

[0047] a) signal peptide;

[0048] b) an antigen-binding domain or portion specific for the TCR-Vβ subunit, preferably an anti-TCRVβ antibody or a functional fragment thereof specific for the TCR-Vβ subunit, more preferably a single-chain variable fragment (scFv) domain of an anti-TCRVβ antibody;

[0049] c) a hinge region or a hinge domain, preferably comprising a transmembrane domain, most preferably a hinge region or a hinge domain comprising a transmembrane domain and a cytoplasmic region;

[0050] d) a primary stimulatory (or signaling) domain; and / or

[0051] e) a co-stimulatory domain, preferably two or more co-stimulatory domains.

[0052] Unexpectedly, the inventors demonstrated that the combination of these components can generate an anti-TCRVβ CAR construct that is highly selective for activity against the homologous TCRVβ family chain and has significant activity against T-cell lymphomas and leukemias in vitro and in vivo.

[0053] Therefore, preferably, the CAR construct comprises a signal peptide. Advantageously, the signal peptide is configured to guide CAR (i.e., fusion protein) to the outer membrane of the effector cell (i.e., T cell) expressing the CAR construct. The signal peptide of the CAR construct can be a natural immunoglobulin gene signal peptide. However, preferably, the signal peptide comprises human CD8α or a fragment or variant thereof. CD8α is highly expressed in human T cells. Therefore, advantageously, when the CAR construct is expressed in human effector T cells, human CD8a provides optimal expression of the CAR construct in human effector cells.

[0054] In one embodiment, the human CD8a signal peptide may have an amino acid sequence represented herein as SEQ ID No: 5 as shown below:

[0055] MALPVTALLLPLALLLHAARP

[0056] [SEQ ID No:5]

[0057] Therefore, preferably, the CAR construct comprises a signal peptide having an amino acid sequence substantially as shown in SEQ ID No: 5 or a fragment or variant thereof.

[0058] In one embodiment, the nucleotide sequence encoding the signal peptide is represented herein as SEQ ID No: 6 as shown below:

[0059] atggctctgcctgtgacagctctgctgctgcctctggccctgctgctgcatgccgccagacct

[0060] [SEQ ID No:6]

[0061] Therefore, preferably, the signal peptide is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 6 or a fragment or variant thereof.

[0062] Preferably, the antigen binding domain or part specific for the TCR-Vβ subunit includes an anti-TCRVβ antibody or a functional fragment thereof specific for the TCR-Vβ subunit. More preferably, the antigen binding domain specific for the TCR-Vβ subunit includes a single-chain variable fragment (scFv) domain of an anti-TCRVβ antibody. Preferably, the antigen binding domain is located at the C-terminus of the signal peptide.

[0063] Those skilled in the art know that scFv is a fusion protein comprising the heavy chain (V H ) and light chain (V L ) variable region. In the context of the present invention, the antibody is an antibody specific for the TCRVβ subunit.

[0064] As described herein, there are 23 different families of TCRVβ chain molecules. It should be understood that the CAR construct of the present invention can target any human TCRVβ chain. TCRβ chain sequences are easily available in public databases. For example, the CAR construct of the present invention can target any TCRVβ listed in Tables 1 and 2, which provide a summary of TCR genes (TRBV) and proteins (TCRVβ) and indicate proteins for which monoclonal antibodies are available or can be developed. Therefore, in one embodiment, the scFv domain of an anti-TCRVβ antibody is selected from any TCRVβ chain gene and protein listed in Tables 1 and 2.

[0065] like Figure 1 As shown in Figure 2, the CAR construct may include a scFv, which may include a VL (variable light chain) sequence and a VH (variable heavy chain) sequence. Preferably, the VL sequence is located upstream of the VH sequence (ie, 5' or N-terminal). However, in some embodiments, the VH sequence may be located upstream of the VL sequence. Preferably, the VH and VL encoding sequences (regardless of direction) are separated by a linker sequence (eg, a G4S linker sequence). Preferably, the linker sequence is flexible.

[0066] TCRVβ1

[0067] In a preferred embodiment, the CAR construct may include an scFv comprising VL and / or VH from an anti-TCRVβ1 antibody. The CAR construct preferably includes an scFv comprising VL and VH from an anti-TCRVβ1 antibody. In one embodiment, the VL and VH sequences may be derived from BL37.2 (i.e., the hybridoma clone name of the anti-TCRVβ1 monoclonal antibody) and include a light chain variable region and a heavy chain variable region for binding to the TCRVβ1 antigen.

[0068] In one embodiment, the light chain of the scFv of the anti-TCRVβ1 antibody may have the amino acid sequence represented herein as SEQ ID No: 7 as shown below:

[0069] DVQMTQSPYNLAASPGESVSINCKASKSINKYLAWYQQKPGKPNKLLIYDGSTLQS GIPSRFSGSGSGTDFTLTIRGLEPEDFGLYYCQQHNEYPPTFGAGTKLELK

[0070] [SEQ ID No:7]

[0071] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 7 or a fragment or variant thereof.

[0072] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ1 antibody is represented herein as SEQ ID No: 8 as shown below:

[0073] GACGTGCAGATGACCCAGAGCCCCTACAACCTGGCCGCCAGCCCCGGCGAGAGCGTGAGCATCAACTGCAAGGCCAGCAAGAGCATCAACAAGTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAGCCCAACAAGCTGCTGATCTACGACGGCAGCACCC TGCAGAGCGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGGGGCCTGGAGCCCGAGGACTTCGGCCTGTACTACTGCCAGCAGCACAACGAGTACCCCCCCACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG

[0074] [SEQ ID No:8]

[0075] Therefore, preferably, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 8 or a fragment or variant thereof.

[0076] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ1 antibody may have the amino acid sequence represented herein as SEQ ID No: 9 as shown below:

[0077] QLQLVQSGPELREPGESVKISCKASGYTFTDYIVHWVKQAPGKGLKWMGWINTYTGTPTYADDFEGRFVFSLEASASTANLQISNLKNEDTATYFCARSWRRGIRGIGFDYWGQGVMVTVSS

[0078] [SEQ ID No:9]

[0079] Therefore, preferably, the CAR construct comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 10 or a fragment or variant thereof.

[0080] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ1 antibody is represented herein as SEQ ID No: 10 as shown below:

[0081] CAGCTGCAGCTGGTGCAGAGCGGCCCCGAGCTGAGGGAGCCCGGCGAGAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACATCGTGCACTGGGTGAAGCAGGCCCCCGGCAAGGGCCTGAAGTGGATGGGCTGGATCAACACCTACACCGGCACCCCCACCTACGCC GACGACTTCGAGGGCAGGTTCGTGTTCAGCCTGGAGGCCAGCGCCAGCACCGCCAACCTGCAGATCAGCAACCTGAAGAACGAGGACACCGCCACCTACTTCTGCGCCAGGAGCTGGAGGAGGGGCATCAGGGGCATCGGCTTCGACTACTGGGGCCAGGGCGTGATGGTGACCGTGAGCAGC

[0082] [SEQ ID No:10]

[0083] Therefore, preferably, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 10 or a fragment or variant thereof.

[0084] TCRVβ2

[0085] In another preferred embodiment, the CAR construct may include an scFv comprising VL and / or VH from an anti-TCRVβ2 antibody. The CAR construct preferably includes an scFv comprising VL and VH from an anti-TCRVβ2 antibody. In one embodiment, the VL and VH sequences may be derived from MPB2D5 (i.e., the hybridoma clone name of the anti-TCRVβ2 monoclonal antibody) and include a light chain variable region and a heavy chain variable region for binding to the TCRVβ2 antigen.

[0086] In one embodiment, the light chain of the scFv of the anti-TCRVβ2 antibody may have the amino acid sequence represented herein as SEQ ID No: 1 as follows:

[0087] DIVLTQSPASLAVSLGQRATISCRASKSVSILGTHLIHWYQQKPGQPPKLLIYAASNL ESGVPARFSGSGSETVFTLNIHPVEEEDAATYFCQQSIEDPWTFGGGTKLGIK

[0088] [SEQ ID No:11]

[0089] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 11 or a fragment or variant thereof.

[0090] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ2 antibody is represented herein as SEQ ID No: 12 as shown below:

[0091] GACATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAGGGCCAGCAAGAGCGTGAGCATCCTGGGCACCCACCTGATCCACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCCAAGCTGCTGATCTACGCCGCtA GCAACCTGGAGAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGAGACCGTGTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTTCTGCCAGCAGAGCATCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGGCATCAAG

[0092] [SEQ ID No:12]

[0093] Therefore, preferably, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 12 or a fragment or variant thereof.

[0094] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ2 antibody may have the amino acid sequence represented herein as SEQ ID No: 13 as shown below:

[0095] EVQLQQSVADLVRPGASLKLSCTASGFNIKSAYMHWVIQRPDQGPECLGRIDPATGKTKYAPKFQAKATITADTSSNTAYLQLSSLTSEDTAIYYCTRSLNWDYGLDYWGQGTSVTVSS

[0096] [SEQ ID No:13]

[0097] Therefore, preferably, the CAR construct comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 13 or a fragment or variant thereof.

[0098] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ1 antibody is represented herein as SEQ ID No: 14 as shown below:

[0099] GAGGTGCAGCTGCAGCAGAGCGTGGCCGACCTGGTGAGGCCCGGCGCCAGCCTGAAGCTGAGCTGCACCGCCAGCGGCTTCCAACATCAAGAGCGCCTACATGCACTGGGTGATCCAGAGGCCCGACCAGGGCCCCGAGTGCCTGGGCAGGATCGACCCCGCCACCGGCAAGACCAAGT ACGCCCCCAAGTTCCAGGCCAAGGCCACCATCACCGCCGACACCAGCAGCAACACCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACACCGCCATCTACTACTGCACCAGGAGCCTGAACTGGGACTACGGCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC

[0100] [SEQ ID No:14]

[0101] Therefore, preferably, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 14 or a fragment or variant thereof.

[0102] TCRVβ9

[0103] In another preferred embodiment, the CAR construct may include an scFv comprising VL and / or VH from an anti-TCRVβ9 antibody. The CAR construct preferably includes an scFv comprising VL and VH from an anti-TCRVβ9 antibody. In one embodiment, the VL and VH sequences may be derived from FIN9 (i.e., the hybridoma clone name of the anti-TCRVβ9 monoclonal antibody) and include a light chain variable region and a heavy chain variable region for binding to the TCRVβ9 antigen.

[0104] In one embodiment, the light chain of the scFv of the anti-TCRVβ9 antibody may have the amino acid sequence represented herein as SEQ ID No: 15 as shown below:

[0105] ETTVTQSPASSLSVATGEKVTIRCISSTDIDDDMNWYQQKSGEPPKLLISEGNTLRPG VPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNMPLTFGAGTKLELK

[0106] [SEQ ID No:15]

[0107] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 15 or a fragment or variant thereof.

[0108] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ9 antibody is represented herein as SEQ ID No: 16 as shown below:

[0109] GAGACCACCGTGACCCAGAGCCCCGCCAGCCTGAGCGTGGCCACCGGCGAGAAGGTGACCATCAGGTGCATCAGCAGCACCGACATCGACGACGACATGAACTGGTACCAGCAGAAGAGCGGCGAGCCCCCCAAGCTGCTGATCAGCGAGGGCAACACCC TGAGGCCCGGCGTGCCCAGCAGGTTCAGCAGCGGCTACGGCACCGACTTCGTGTTCACCATCGAGAACATGCTGAGCGAGGACGTGGCCGACTACTACTGCCTGCAGAGCGACAACATGCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG

[0110] [SEQ ID No:16]

[0111] Therefore, preferably, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 16 or a fragment or variant thereof.

[0112] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ9 antibody may have the amino acid sequence represented herein as SEQ ID No: 17 as shown below:

[0113] EVQLQQSVAELVRPGASVKLSCTASGFNIKNTFMHWVKQRPEQGLEWIGRIDPTNGYTKFAPKFQGKATLTAVTSSNTVYLQLSSLTSEDTAIYYCAHDYDAPWFAYWGQGTLVIVSA

[0114] [SEQ ID No:17]

[0115] Therefore, preferably, the CAR construct comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 17 or a fragment or variant thereof.

[0116] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ9 antibody is represented herein as SEQ ID No: 18 as shown below:

[0117] GAGGTGCAGCTGCAGCAGAGCGTGGCCGAGCTGGTGAGGCCCGGCGCCAGCGTGAAGCTGAGCTGCACCGCCAGCGGCTTCCAACATCAAGAACACCTTCATGCACTGGGTGAAGCAGAGGCCCGAGCAGGGCCTGGAGTGGATCGGCAGGATCGACCCACCAACGGCTACACCAAG TTCGCCCCCAAGTTCCAGGGCAAGGCCACCCTGACCGCCGTGACCAGCAGCAACACCGTGTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACACCGCCATCTACTGCGCCCACGACTACGACGCCCCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGATCGTGAGCGCC

[0118] [SEQ ID No:18]

[0119] Therefore, preferably, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 18 or a fragment or variant thereof.

[0120] TCRVβ11

[0121] In another preferred embodiment, the CAR construct may include an scFv comprising VL and / or VH from an anti-TCRVβ11 antibody. The CAR construct preferably includes an scFv comprising VL and VH from an anti-TCRVβ11 antibody. In one embodiment, the VL and VH sequences may be derived from C21 (i.e., the hybridoma clone name of the anti-TCRVβ11 monoclonal antibody) and include a light chain variable region and a heavy chain variable region for binding to the TCRVβ11 antigen.

[0122] In one embodiment, the light chain of the scFv of the anti-TCRVβ11 antibody may have the amino acid sequence represented herein as SEQ ID No: 19 as shown below:

[0123] DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGGGTRLEIK

[0124] [SEQ ID No: 19]

[0125] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 19 or a fragment or variant thereof.

[0126] In one embodiment, the nucleotide sequence encoding the VL chain of the scFv of the anti-TCRVβ11 antibody is represented herein as SEQ ID No: 20 as shown below:

[0127] GACATTAAGATGACCCAGTCCCCCTCCTCCATGTATGCCAGCCTCGGCGAGAGAGTCACCATCACATGCAAGGCCAGCCAAGACATCAACAGCTACCTCAGCTGGTTTCCAGCAGAAAGCCGGCAAGAGCCCCAAGACACTGATCTATAGGGCTAATAGAC TGGTGGACGGCGTGCCTAGCAGATTTTCCGGCAGCGGCAGCGGCCAAGACTATTCTCTGACCATCAGCTCTCTGGAGTACGAGGACATGGGAATCTACTACTGTCTGCAGTACGACGAGTTCCCCTTCACCTTCGGAGGCGGCACAAGACTGGAAATCAAA

[0128] [SEQ ID No:20]

[0129] Therefore, preferably, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 20 or a fragment or variant thereof.

[0130] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ11 antibody may have the amino acid sequence represented herein as SEQ ID No: 21 as shown below:

[0131] QVQLQQSGPEVVRPGVSVKISCKGSGYRFTDSAMHWVKQSHAKSLEWIGVISSYNGNTNYNQKFKGKATMTVDKSSSTAYMELARMTSEDSAIYYCARSRDAMDYWGQGTSVTVSS

[0132] [SEQ ID No:21]

[0133] Therefore, preferably, the CAR construct comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 21 or a fragment or variant thereof.

[0134] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ11 antibody is represented herein as SEQ ID No: 22 as shown below:

[0135] CAAGTGCAGCTCCAGCAGTCCGGACCCGAGGTGGTGAGGCCCGGCGTGAGCGTGAAGATCAGCTGCAAGGGCAGCGGCTATAGGTTCACCGACTCCGCCATGCACTGGGTGAAGCAGTCCCATGCCAAGAGCCTCGAGTGGATCGGCGTGATCAGCAGCTACAACGGCAACACC AACTACAACCAGAAGTTCAAGGGCAAGGCCACATGACCGTGGACAAGAGCAGCTCCACCGCCTACATGGAGCTGGCCAGAATGACCAGCGAGGATAGCGCCATCTACTACTGTGCTAGGTCTAGAGACGCCATGGACTACTGGGGCCAAGGCACATCCGTGACCGTGAGCTCC

[0136] [SEQ ID No:22]

[0137] Therefore, preferably, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 22 or a fragment or variant thereof.

[0138] Preferably, the VH (e.g., SEQ ID No: 9, 13, 17 or 21) and VL (e.g., SEQ ID No: 7, 11, 15 or 19) sequences are separated by a linker sequence in either direction. In one embodiment, the linker sequence may comprise at least one G4S linker sequence, which may be represented herein as SEQ ID No: 23 as shown below:

[0139] GGGGS

[0140] [SEQ ID No:23]

[0141] Therefore, preferably, the CAR construct comprises a linker sequence comprising an amino acid sequence substantially as shown in SEQ ID No: 23 or a fragment or variant thereof.

[0142] In other embodiments, the linker sequence may comprise a plurality of repeated G4S linker sequences. For example, the linker sequence may comprise 2 or 3 repeated G4S linker sequences (i.e., 2 x G4S linker sequences). 4 S or 3x G 4 S).

[0143] The CAR construct preferably includes a hinge domain. Preferably, the hinge domain includes: (i) an extracellular domain or a portion thereof; (ii) a transmembrane (TM) domain or a portion thereof; and / or (iii) a cytoplasmic domain or a portion thereof. Most preferably, the hinge domain includes: (i) an extracellular domain or a portion thereof; (ii) a transmembrane (TM) domain or a portion thereof; and (iii) a cytoplasmic domain or a portion thereof. Advantageously, the hinge domain is configured for CAR display and anchoring on CAR-T cells. Preferably, the hinge domain is located at the C-terminus of the antigen binding domain, more preferably at the C-terminus of the VH chain.

[0144] Preferably, the hinge domain comprises a CD8α sequence or a portion thereof, more preferably a human CD8α sequence or a portion thereof. Advantageously, a longer hinge derived from CD8α can significantly reduce the toxicity of the CAR construct compared to a shorter hinge derived from CD8α. Therefore, preferably, in one embodiment, the hinge domain comprises or is substantially composed of a full-length human CD8α or a portion thereof.

[0145] In one embodiment, the amino acid sequence of human CD8α is represented herein as SEQ ID No: 51 as shown below:

[0146] MALPPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSA LSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

[0147] [SEQ ID No:51]

[0148] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which comprises at least a portion of the extracellular domain. In one embodiment, the amino acid sequence of the extracellular domain of human CD8α or a portion thereof is represented herein as SEQ ID No: 24 as shown below:

[0149] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0150] [SEQ ID No:24]

[0151] Therefore, preferably, the CAR construct comprises a hinge domain comprising an amino acid sequence substantially as shown in SEQ ID No: 24 or a fragment or variant thereof.

[0152] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which comprises a full transmembrane helical domain. In one embodiment, the amino acid sequence of the full transmembrane domain of human CD8α is represented herein as SEQ ID No: 49 as shown below:

[0153] IYIWAPLAGTCGVLLLSLVIT

[0154] [SEQ ID No:49]

[0155] Thus, preferably, the CAR construct comprises or consists of a hinge domain comprising an amino acid sequence substantially as shown in SEQ ID No: 49 or a fragment or variant thereof.

[0156] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which comprises at least a portion of the cytoplasmic domain. In one embodiment, the amino acid sequence of the human CD8α cytoplasmic domain or a portion thereof is represented herein as SEQ ID No: 50 as shown below:

[0157] LYCNHRN

[0158] [SEQ ID No:50]

[0159] Thus, preferably, the CAR construct comprises or consists of a hinge domain comprising an amino acid sequence substantially as shown in SEQ ID No: 50 or a fragment or variant thereof.

[0160] In a preferred embodiment, the CAR construct comprises a hinge domain derived from human CD8α. Preferably, the hinge comprises or consists of amino acids 128-182 (i.e., the extracellular domain) of human CD8α as shown in SEQ ID No: 51. Preferably, the hinge comprising a portion of the extracellular domain of human CD8α, a full transmembrane domain of human CD8α, and a portion of the cytoplasmic domain of human CD8α is defined by amino acids 128-210 of human CD8α, which may have an amino acid sequence represented herein as SEQ ID No: 25 as shown below:

[0161] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

[0162] [SEQ ID No:25]

[0163] Therefore, preferably, the CAR construct comprises or consists of a hinge domain having an amino acid sequence substantially as shown in SEQ ID No: 25 or a fragment or variant thereof.

[0164] Advantageously, SEQ ID No: 25 comprises 55 amino acids from the extracellular domain (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD-SEQ ID No: 24), a full transmembrane helical domain (IYIWAPLAGTCGVLLLSLVIT-SEQ ID No: 49) and 7 amino acids from the cytoplasmic domain (LYCNHRN-SEQ ID No: 50). This structure advantageously reduces the risk of cytokine release syndrome and neurotoxicity (these are side effects commonly associated with CAR-T therapy) of the CAR construct without negatively affecting the efficacy of the construct.

[0165] In one embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which hinge domain comprises the extracellular domain of human CD8α, which is defined by amino acids 138-182 of human CD8α as shown in SEQ ID No:51.

[0166] Preferably, the hinge derived from CD8α comprising the extracellular domain of human CD8α is at least 1, 2 or 3 amino acids longer at the N-terminus than amino acids 138-182 of human CD8α as shown in SEQ ID No: 51. Therefore, preferably, the hinge derived from CD8α comprises the extracellular domain of human CD8α defined by amino acids 137-182, 136-182 or 135-182 of human CD8α as shown in SEQ ID No: 51.

[0167] However, more preferably, the hinge derived from CD8α comprising the extracellular domain of human CD8α is at least 4, 5 or 6 amino acids longer at the N-terminus than amino acids 138-182 of human CD8α as shown in SEQ ID No: 51. Therefore, more preferably, the hinge derived from CD8α comprises the extracellular domain of human CD8α defined by amino acids 134-182, 133-182 or 132-182 of human CD8α as shown in SEQ ID No: 51.

[0168] Most preferably, the hinge derived from CD8α comprising the extracellular domain of human CD8α is at least 7, 8, 9 or 10 amino acids longer at the N-terminus than amino acids 138-182 of human CD8α as shown in SEQ ID No: 51. Therefore, most preferably, the hinge derived from CD8α comprises the extracellular domain of human CD8α defined by amino acids 131-182, 130-182, 129-182 or 128-182 of human CD8α as shown in SEQ ID No: 51.

[0169] In one embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which hinge domain comprises the transmembrane domain of human CD8α defined by amino acids 183-203 of human CD8α as shown in SEQ ID No:51.

[0170] In another embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, which hinge domain comprises the cytoplasmic domain of human CD8α defined by amino acids 204-206 of human CD8α as shown in SEQ ID No:51.

[0171] Preferably, the hinge derived from CD8α comprising the cytoplasmic domain of human CD8α is at least 1 amino acid longer at the C-terminus than amino acids 204-206 of human CD8α as shown in SEQ ID No: 51. Therefore, preferably, the hinge derived from CD8α comprises the cytoplasmic domain of human CD8α defined by amino acids 204-207 of human CD8α as shown in SEQ ID No: 51.

[0172] More preferably, the hinge derived from CD8α comprising the cytoplasmic domain of human CD8α is at least 2 amino acids longer at the C-terminus than amino acids 204-206 of human CD8α as shown in SEQ ID No: 51. Therefore, more preferably, the hinge derived from CD8α comprises the cytoplasmic domain of human CD8α defined by amino acids 204-208 of human CD8α as shown in SEQ ID No: 51.

[0173] Still more preferably, the hinge derived from CD8α comprising the cytoplasmic domain of human CD8α is at least 3 amino acids longer at the C-terminus than amino acids 204-206 of human CD8α as shown in SEQ ID No: 51. Therefore, still more preferably, the hinge derived from CD8α comprises the cytoplasmic domain of human CD8α defined by amino acids 204-209 of human CD8α as shown in SEQ ID No: 51.

[0174] Most preferably, the hinge derived from CD8α comprising the cytoplasmic domain of human CD8α is at least 4 amino acids longer at the C-terminus compared to amino acids 204-206 of human CD8α as shown in SEQ ID No: 51. Therefore, most preferably, the hinge derived from CD8α comprises the cytoplasmic domain of human CD8α defined by amino acids 204-210 of human CD8α as shown in SEQ ID No: 51.

[0175] In one embodiment, the nucleotide sequence encoding the hinge domain is represented herein as SEQ ID No: 26 as shown below:

[0176] ttcgtgcctgtgtttctgcctgccaagcccaccacaacccctgcccctagacctcctacacccgcccctacaatcgccagccagcctctgtctctgaggcccgaggcttgtagacctgctgctg gcggagccgtgcacaccagaggactggatttcgcctgcgacatctacatctgggcccctctggccggcacatgtggcgtgctgctgctgagcctcgtgatcaccctgtactgcaaccaccggaac

[0177] [SEQ ID No:26]

[0178] Thus, preferably, the construct comprises a hinge domain encoded by a nucleotide sequence substantially as shown in SEQ ID No: 26 or a fragment or variant thereof.

[0179] The CAR construct preferably comprises an intracellular domain comprising a co-stimulatory (or signaling) domain of a major stimulation (or signaling) CD3 ζ chain and / or CD28, more preferably a co-stimulatory / signaling domain of CD28 and a stimulating CD3 ζ chain. It should be understood that these components form the basis of the second generation CAR and are required to trigger the intracellular signaling pathway. Preferably, the intracellular domain is located at the 3' end of the sequence encoding the hinge domain. The co-stimulatory domain of CD28 can be the N-terminus of the CD3 ζ chain. Advantageously, the CD28 co-stimulatory domain significantly improves the efficacy of CD19 CAR T cells in patients with B cell lymphoma and B cell acute leukemia, and makes TCRVβCAR have significant efficacy in vitro and in vivo against T cell lymphoma and leukemia.

[0180] In another embodiment, the CAR construct may comprise 1 or 2 co-stimulatory domains, which may be selected from CD28, a 4-1BB signaling domain, and an OX40 signaling domain.

[0181] One embodiment of a 4-1BB signaling domain may have an amino acid sequence represented herein as SEQ ID No: 46 as follows:

[0182] RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0183] [SEQ ID No:46]

[0184] Therefore, preferably, the construct comprises a nucleotide sequence encoding an amino acid sequence substantially as shown in SEQ ID No: 46 or a fragment or variant thereof.

[0185] In one embodiment, the 4-1BB signaling domain may be encoded by the nucleic acid sequence represented herein as SEQ ID No: 47 as follows:

[0186] CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0187] [SEQ ID No:47]

[0188] Therefore, preferably, the construct comprises a nucleotide sequence substantially as shown in SEQ ID No: 47 or a fragment or variant thereof.

[0189] Therefore, in one embodiment, the costimulatory domain of CD28 is represented herein as SEQ ID No: 27 as shown below:

[0190] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0191] [SEQ ID No:27]

[0192] Therefore, preferably, the CAR construct comprises a costimulatory domain of CD28 having an amino acid sequence substantially as shown in SEQ ID No: 27 or a fragment or variant thereof.

[0193] In one embodiment, the nucleotide sequence encoding the costimulatory domain of CD28 is represented herein as SEQ ID No: 28 as shown below:

[0194] agaagcaagcggagccggctgctgcacagcgactacatgaacatgacccccagacggcctggccccaccagaaagcactacca gccttacgcccctcccagagacttcgccgcctaccggtcc

[0195] [SEQ ID No:28]

[0196] Therefore, preferably, the costimulatory domain of CD28 is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 28 or a fragment or variant thereof.

[0197] In a preferred embodiment, the CAR construct further comprises a stimulatory protein CD3ζ. In one embodiment, the stimulatory protein CD3ζ may have an amino acid sequence represented herein as SEQ ID No: 29 as shown below:

[0198] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0199] [SEQ ID No:29]

[0200] Therefore, preferably, the CAR construct comprises the stimulatory protein CD3ζ having an amino acid sequence substantially as shown in SEQ ID No: 29 or a fragment or variant thereof.

[0201] In one embodiment, the nucleotide sequence encoding the stimulatory protein CD3ζ is represented herein as SEQ ID No: 30 as shown below:

[0202] agagtgaagttcagcagaagcgccgacgcccctgcctatcagcagggccagaaccagctgtacaacgagctgaacctgggcagacgggaagagtacgatgtgctggacaaaagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaa ggcctgtacaatgaactgcagaaagataagatggcgaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

[0203] [SEQ ID No:30]

[0204] Therefore, preferably, the stimulatory protein CD3ζ is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 30 or a fragment or variant thereof.

[0205] Therefore, in a preferred embodiment, the intracellular domain of the CAR construct of the first aspect comprises the co-stimulatory domain of CD28 and the CD3ζ stimulatory domain.

[0206] It should be understood that the position of each component in the CAR constructs described herein is interchangeable. Figure 1 A schematic diagram of an exemplary embodiment of an anti-TCRVβ CAR construct is provided.

[0207] Therefore, in a preferred embodiment, the CAR construct comprises each element in the following order: 5' / N-terminal variable light chain of anti-TCRVβ antibody-variable heavy chain of anti-TCRVβ antibody-hinge-comprising the co-stimulatory domain of CD28-3' / C-terminal stimulatory protein CD3ζ. The N-terminus and C-terminus (as well as 5' and 3') are used to indicate that the feature is located upstream or downstream of the construct (protein or DNA, respectively), and it is not intended to indicate that the feature must be a terminal feature.

[0208] More preferably, the CAR construct comprises 5' / N-terminal signal peptide-variable light chain of anti-TCRVβ antibody-linker-variable heavy chain of anti-TCRVβ antibody-hinge-co-stimulatory domain containing CD28-3' / C-terminal stimulatory protein CD3ζ.

[0209] "BL37.2 CAR"

[0210] In a preferred embodiment, the anti-TCRVβCAR construct is an anti-TCRVβ1CAR (represented herein as "BL37.2 CAR"). Preferably, the anti-TCRVβ1CAR comprises 2x G 4 In one embodiment, the anti-TCRVβ1CAR (with 2x G 4 S linker) has an amino acid sequence represented herein as SEQ ID No: 31 as follows:

[0211] MALPPVTALLLPLALLLHAARPDVQMTQSPYNLAASPGESVSINCKASKSINKYLAWYQQKPGKPNKLLIYDGSTLQSGIPSRFSGSGSGTDFTLTIRGLEPEDFGLYYCQQHNEYPPTFGAGTK LELKGGGGSGGGGSQLQLVQSGPELREPGESVKISCKASGYTFTDYIVHWVKQAPGKGLKWMGWINTYTGTPTYADDFEGRFVFSLEASASTANLQISNLKNEDTATYFCARSWRRGIRGIGFD YWGQGVMVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0212] [SEQ ID No:31]

[0213] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 31 or a fragment or variant thereof.

[0214] In one embodiment, the encoding anti-TCRVβ1 CAR (with 2x G 4 The nucleotide sequence of the S linker) is represented herein as SEQ ID No:32 as shown below:

[0215]

[0216] [SEQ ID No:32]

[0217] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 32 or a fragment or variant thereof.

[0218] In another preferred embodiment, the anti-TCRVβ1 CAR construct (referred to as "BL37.2 CAR") comprises 3x G 4 In one embodiment, the anti-TCRVβ1CAR (with 3x G 4 S linker) may have an amino acid sequence represented herein as SEQ ID No: 33 as follows:

[0219] MALPPVTALLLPLALLLHAARPDVQMTQSPYNLAASPGESVSINCKASKSINKYLAWYQQKPGKPNKLLIYDGSTLQSGIPSRFSGSGSGTDFTLTIRGLEPEDFGLYYCQQHNEYPPTFGAGTKL ELKGGGGSGGGGSGGGGSQLQLVQSGPELREPGESVKISCKASGYTFTDYIVHWVKQAPGKGLKWMGWINTYTGTPTYADDFEGRFVFSLEASASTANLQISNLKNEDTATYFCARSWRRGIRGI GFDYWGQGVMVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0220] [SEQ ID No:33]

[0221] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 33 or a fragment or variant thereof.

[0222] In one embodiment, the encoding anti-TCRVβ1 CAR (with 3x G 4The nucleotide sequence of the S linker) is represented herein as SEQ ID No: 34 as shown below:

[0223]

[0224] [SEQ ID No:34]

[0225] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 34 or a fragment or variant thereof.

[0226] "MPB2D5 CAR"

[0227] In another preferred embodiment, the anti-TCRVβCAR construct is an anti-TCRVβ2CAR (referred to as "MPB2D5CAR"). Preferably, the anti-TCRVβ2CAR comprises 2x G 4 In one embodiment, the anti-TCRVβ2CAR (with 2x G 4 S linker) has an amino acid sequence represented herein as SEQ ID No: 35 as follows:

[0228] MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGQRATISCRASKSVSILGTHLIHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSETVFTLNIHPVEEEDAATYFCQQSIEDPWTFG GGTKLGIKGGGGSGGGGSEVQLQQSVADLVRPGASLKLSCTASGFNIKSAYMHWVIQRPDQGPECLGRIDPATGKTKYAPKFQAKATITADTSSNTAYLQLSSLTSEDTAIYYCTRSLNWDYGL DYWGQGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0229] [SEQ ID No:35]

[0230] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 35 or a fragment or variant thereof.

[0231] In one embodiment, the encoding anti-TCRVβ2 CAR (with 2x G 4 The nucleotide sequence of the S linker) is represented herein as SEQ ID No:36 as shown below:

[0232]

[0233] [SEQ ID No:36]

[0234] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 36 or a fragment or variant thereof.

[0235] In another preferred embodiment, the anti-TCRVβ2 CAR construct (referred to as "MPB2D5 CAR") comprises 3x G 4 In one embodiment, the anti-TCRVβ2CAR (with 3x G 4 S linker) may have an amino acid sequence represented herein as SEQ ID No: 37 as follows:

[0236] MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGQRATISCRASKSVSILGTHLIHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSETVFTLNIHPVEEEDAATYFCQQSIEDPWTFGG GTKLGIKGGGGSGGGGSGGGGSEVQLQQSVADLVRPGASLKLSCTASGFNIKSAYMHWVIQRPDQGPECLGRIDPATGKTKYAPKFQAKATITADTSSNTAYLQLSSLTSEDTAIYYCTRSLNWDY GLDYWGQGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0237] [SEQ ID No:37]

[0238] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 37 or a fragment or variant thereof.

[0239] In one embodiment, the encoding anti-TCRVβ2 CAR (with 3x G 4The nucleotide sequence of the S linker) is represented herein as SEQ ID No: 38 as shown below:

[0240]

[0241] [SEQ ID No:38]

[0242] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 38 or a fragment or variant thereof.

[0243] "FIN9 CAR"

[0244] In a preferred embodiment, the anti-TCRVβCAR construct is an anti-TCRVβ9CAR (referred to as "FIN9CAR"). Preferably, the anti-TCRVβ9CAR comprises 2x G 4 S linker. In one embodiment, the anti-TCRVβ9CAR construct has an amino acid sequence represented herein as SEQ ID No: 39 as follows:

[0245] MALPVTALLLPLALLLHAARPETTVTQSPASLSVATGEKVTIRCISSTDIDDDMNWYQQKSGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNMPLTFGAGT KLELKGGGGSGGGGSEVQLQQSVAELVRPGASVKLSCTASGFNIKNTFMHWVKQRPEQGLEWIGRIDPTNGYTKFAPKFQGKATLTAVTSSNTVYLQLSSLTSEDTAIYYCAHDYDAPWFAYW GQGTLVIVSAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0246] [SEQ ID No:39]

[0247] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 39 or a fragment or variant thereof.

[0248] In one embodiment, the encoding anti-TCRVβ9 CAR (with 2x G 4 The nucleotide sequence of the S linker) is represented herein as SEQ ID No:40 as shown below:

[0249]

[0250] [SEQ ID No:40]

[0251] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 40 or a fragment or variant thereof.

[0252] In another preferred embodiment, the anti-TCRVβ9 CAR construct (referred to as "FIN9 CAR") comprises 3x G 4 In one embodiment, the anti-TCRVβ9CAR (with 3x G 4 S linker) may have an amino acid sequence represented herein as SEQ ID No:41 as follows:

[0253] MALPVTALLLPLALLLHAARPETTVTQSPASLSVATGEKVTIRCISSTDIDDDMNWYQQKSGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNMPLTFGAGTK LELKGGGGSGGGGSGGGGSEVQLQQSVAELVRPGASVKLSCTASGFNIKNTFMHWVKQRPEQGLEWIGRIDPTNGYTKFAPKFQGKATLTAVTSSNTVYLQLSSLTSEDTAIYYCAHDYDAPWF AYWGQGTLVIVSAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0254] [SEQ ID No:41]

[0255] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 41 or a fragment or variant thereof.

[0256] In one embodiment, the encoding anti-TCRVβ9 CAR (with 3x G 4The nucleotide sequence of S) is represented herein as SEQ ID No:42 as shown below:

[0257]

[0258] [SEQ ID No:42]

[0259] Therefore, preferably, the construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 42 or a fragment or variant thereof.

[0260] "C21 CAR"

[0261] In another preferred embodiment, the anti-TCRVβCAR construct is an anti-TCRVβ11CAR (referred to as "C21CAR"). Preferably, the anti-TCRVβ11CAR comprises 2x G 4 In one embodiment, the anti-TCRVβ11 CAR construct (with 2xG 4 S linker) may have an amino acid sequence represented herein as SEQ ID No:43 as follows:

[0262] MALPVTALLLPLALLLHAARPDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGGG TRLEIKGGGGSGGGGSQVQLQQSGPEVVRPGVSVKISCKGSGYRFTDSAMHWVKQSHAKSLEWIGVISSYNGNTNYNQKFKGKATMTVDKSSSTAYMELARMTSEDSAIYYCARSRDAMDYWG QGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0263] [SEQ ID No:43]

[0264] Therefore, preferably, the CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 43 or a fragment or variant thereof.

[0265] In one embodiment, the encoding anti-TCRVβ11 CAR (with 2x G 4 The nucleotide sequence of the S linker) is represented herein as SEQ ID No:52 as shown below:

[0266]

[0267] [SEQ ID No:52]

[0268] Therefore, preferably, the construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 52 or a fragment or variant thereof.

[0269] In another preferred embodiment, the anti-TCRVβ11 CAR construct (referred to as "C21 CAR") comprises 3x G 4 In one embodiment, the anti-TCRVβ11 CAR construct (with 3x G 4 S linker) may have an amino acid sequence represented herein as SEQ ID No: 44 as follows:

[0270] MALPVTALLLPLALLLHAARPDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPFTFGGGT RLEIKGGGGSGGGGSGGGGSQVQLQQSGPEVVRPGVSVKISCKGSGYRFTDSAMHWVKQSHAKSLEWIGVISSYNGNTNYNQKFKGKATMTVDKSSSTAYMELARMTSEDSAIYYCARSRDAMD YWGQGTSVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0271] [SEQ ID No:44]

[0272] Therefore, preferably, the CAR construct has an amino acid sequence substantially as shown in SEQ ID No: 44 or a fragment or variant thereof.

[0273] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ11 CAR (with 3x G4S linker) is represented herein as SEQ ID No: 45 as shown below:

[0274]

[0275] [SEQ ID No:45]

[0276] Therefore, preferably, the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 45 or a fragment or variant thereof.

[0277] In the second aspect, a nucleic acid encoding the CAR construct of the first aspect is provided.

[0278] Preferably, the nucleic acid comprises a promoter operably linked to a sequence encoding a CAR construct. The promoter is preferably located at the 5' end of the sequence encoding the signal peptide. The promoter drives the expression of the CAR construct in the host cell.

[0279] The promoter can be any suitable promoter, including a constitutive promoter, an activatable promoter, an inducible promoter or a tissue-specific promoter. A constitutive promoter causes a heterologous gene (also expressed as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters considered herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1a), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoters (PGK), simian virus 40 early promoters (SV40) and chicken beta-actin promoters (CAGG) coupled to CMV early enhancers. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by more than one condition, such as physical conditions, the microenvironment of engineered immune effector cells or the physiological state of engineered immune effector cells, inducers (i.e., inducers) or combinations thereof. In some embodiments, the induction conditions will not induce the expression of endogenous genes in engineered mammalian cells and / or subjects receiving the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of an inducer, radiation (eg, ionizing radiation, light), temperature (eg, heat), redox state, tumor environment, and activation state of the engineered mammalian cell.

[0280] In one embodiment, the promoter may be the PGK promoter (EMBL NO: A19297.1). In one embodiment, the PGK promoter is represented herein as SEQ ID No: 48 as shown below:

[0281] GGGTAGGGGAGGCCTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCACTAG TCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCG GGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATTCTCCGGGCCTTTCG

[0282] [SEQ ID No:48]

[0283] Therefore, preferably, the promoter may comprise a nucleotide sequence substantially as shown in SEQ ID No: 48 or a fragment or variant thereof.

[0284] In a preferred embodiment, the nucleic acid of the second aspect is selected from any nucleic acid sequence described herein.

[0285] In a third aspect, an expression vector or plasmid encoding the CAR construct of the first aspect, or an expression vector or plasmid comprising the nucleic acid of the second aspect is provided.

[0286] Preferably, the vector is recombinant. The vector is preferably a viral vector, more preferably a retroviral vector, and even more preferably a lentiviral vector. Figure 4-7 A diagram of the vector plasmid structure of exemplary anti-TCRVβCAR constructs tested in the present invention is shown.

[0287] Preferably, the vector comprises a left (ie N-terminal) and / or right (ie C-terminal) long terminal repeat (LTR). Preferably, each LTR is located at the N-terminal and / or C-terminal of the construct.

[0288] In a preferred embodiment, the vector comprises 5' / N-terminal LTR-promoter-variable light chain of the scFv domain of anti-TCRVβ antibody-linker-variable heavy chain of the scFv domain of anti-TCRVβ antibody-hinge-co-stimulatory domain containing CD28-stimulatory protein CD3ζ-3' / C-terminal LTR.

[0289] Preferably, the hinge comprises a CD8 alpha hinge, and optionally a transmembrane and / or cytoplasmic domain.

[0290] In a fourth aspect, an effector cell expressing the CAR construct of the first aspect, an effector cell comprising the nucleic acid of the second aspect, or an effector cell comprising the vector of the third aspect is provided.

[0291] The vector may be a lentiviral vector. The vector may be a retroviral vector.

[0292] Preferably, the effector cell is a normal or conventional αβ T cell, or an innate lymphocyte, such as an invariant natural killer T (iNKT) cell, a γδ T cell, or a NK cell.

[0293] Those skilled in the art know that conventional αβT cells are one of the two main types of lymphocytes (B cells are the second type), which determine the specificity of the immune response to antigens (foreign substances) in the body. T cells coordinate multiple aspects of adaptive immunity throughout life, including responses to pathogens, allergens, and tumors, by producing cytokines and effector molecules. In humans, T cells simultaneously control multiple insults throughout the body and maintain immune homeostasis for decades.

[0294] Thus, in one embodiment, the effector cells of the invention are conventional T cells.

[0295] "Conventional T cells" can be defined as T lymphocytes that express the αβ T cell receptor (TCR) and the co-receptors CD4 or CD8 and are present in peripheral blood, lymph nodes and tissues.

[0296] However, in another preferred embodiment, the effector cells are iNKT cells.

[0297] iNKT cells are a subset of immunoregulatory and effector T cells that account for less than 0.1% of the total human T cell population.

[0298] There are some structural and functional differences between iNKT cells and conventional T cells. Specifically, iNKT cells express a constant Va24Ja18 chain that is almost always paired with the same TCR Vβ11 diversified chain. In addition, iNKT cells are also restricted by the non-polymorphic HLA class I-like molecule CD1d, which presents endogenous or exogenous glycolipid or phospholipid ligands to the iTCR. Compared with conventional T cells, which are restricted by highly polymorphic MHC-presented peptides, iNKT cells need to express CD1d on thymocytes to be selected and activated. In contrast, conventional T cells need to express MHC molecules on thymic epithelial cells to be selected and activated.

[0299] iNKT cells provide effective immune responses against infectious pathogens, tumors, allogeneic and autoreactivity, and atherosclerosis. Multiple preclinical studies have shown that adoptively transferred donor iNKT cells can prevent or even eliminate developed experimental acute graft-versus-host disease (aGVHD), an alloreactive phenomenon that occurs in allogeneic hematopoietic stem cell transplantation. aGVHD is primarily caused by donor alloreactive T cells that are activated by differences in major or minor histocompatibility antigens between the donor and recipient. In line with preclinical evidence, multiple clinical observational studies have shown that transferring higher doses or frequencies of donor iNKT cells to recipients with peripheral blood stem cell transplantation can significantly protect them from aGVHD without compromising the graft-versus-tumor effect.

[0300] In humans, iNKT cells vary quantitatively and qualitatively in different types of tumors, including blood cancers such as multiple myeloma, while infiltration of the tumor bed by iNKT cells appears to confer a favorable prognosis in colorectal cancer.

[0301] Much of the antitumor effect of iNKT cells depends on their ability to lyse CD1d-expressing tumors directly via perforin / granzyme and other cell death pathways, indirectly via secretion of large amounts of (interferon-γ) IFNγ, and / or secondary activation of NK cell- or conventional T cell-dependent antitumor responses.

[0302] It will be appreciated that the effector cells of the fourth aspect are produced by transducing T cells or iNKT cells with a nucleic acid or vector encoding a CAR construct.

[0303] Therefore, in the fifth aspect, a method for producing effector cells expressing anti-TCRVβCAR is provided, the method comprising transducing the effector cells with the nucleic acid of the second aspect or the vector of the third aspect, so that the effector cells express the anti-TCRVβCAR.

[0304] Preferably, the effector cell is a normal or conventional αβ T cell, or an innate lymphocyte, such as an invariant natural killer T (iNKT) cell, a γδ T cell, or a NK cell.

[0305] Preferably, the method comprises an initial step of isolating effector cells from peripheral blood cells (PBC). Preferably, the effector cells are activated with one or both of CD3 and CD28 antibodies. Preferably, the effector cells are activated with an interleukin. The interleukin may be IL-15. In a preferred embodiment, the effector cells are activated with CD3 and CD28 antibodies and IL-15.

[0306] In a preferred embodiment, the nucleic acid and / or vector encoding the anti-TCRVβ CAR may include any nucleic acid or vector described herein.

[0307] In the sixth aspect, a pharmaceutical composition is provided, which comprises a therapeutically effective amount of the CAR construct of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect or the effector cell of the fourth aspect, and a pharmaceutically acceptable excipient.

[0308] Preferably, the effector cells are normal (ie conventional αβ T cells) or innate lymphocytes, such as invariant natural killer T (iNKT) cells, γδ T cells or NK cells.

[0309] Preferably, the pharmaceutical composition comprises a plurality of effector cells of the invention, preferably T cells or iNKT cells. For example, the composition may comprise at least 100, 1000 or 10,000 effector cells. Preferably, the composition comprises at least 100,000, at least 1,000,000 or at least 10,000,000 effector cells.

[0310] In the seventh aspect, the use of the CAR construct of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the effector cell of the fourth aspect or the pharmaceutical composition of the sixth aspect for treatment or diagnosis is provided.

[0311] In the eighth aspect, the CAR construct of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the effector cell of the fourth aspect, or the pharmaceutical composition of the sixth aspect is provided for use in (i) immunotherapy; (ii) treating, preventing or improving cancer; (iii) treating, preventing or improving autoimmune diseases; or (iv) treating, preventing or improving any disease characterized by the presence of pathogenic T cells.

[0312] In the ninth aspect, the present invention provides a method for (i) treating, preventing or improving a subject's disease with immunotherapy; (ii) treating, preventing or improving cancer; (iii) treating, preventing or improving a subject's autoimmune disease; or (iv) treating, preventing or improving any disease characterized by the presence of pathogenic T cells, the method comprising administering or having administered a therapeutically effective amount of the CAR construct of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the effector cell of the fourth aspect, or the pharmaceutical composition of the sixth aspect to a patient in need of such treatment.

[0313] In one embodiment of the eighth or ninth aspect of the present invention, the CAR construct, effector cell or pharmaceutical composition is used to treat, prevent or improve cancer. Preferably, the cancer is a T cell malignancy, which can be a solid tumor or a liquid tumor.

[0314] According to the WHO 2022 classification of hematolymphatic tumors, T cell tumors can be precursor T cell tumors, such as T lymphoblastic leukemia / lymphoma, mature T cell tumors, or peripheral T cell lymphoma (PTCL). Overall, with current treatments, the 5-year survival rate is 35-40%. According to the WHO 2022 classification, mature T cell tumors include a diverse group of rare and aggressive diseases in which the patient's T cells become cancerous. They are divided into three categories, namely, nodal, extranodal, and leukemic, each of which is included in the present invention.

[0315] The mature T-cell neoplasm can be a TCL subtype selected from the following: mature T-cell leukemia; primary cutaneous T-cell lymphoproliferation and lymphoma; intestinal T-cell lymphoproliferation and lymphoma; hepatosplenic T-cell lymphoma; anaplastic large cell lymphoma; nodal T follicular helper (TFH) cell lymphoma; peripheral T-cell lymphoma, NOS; EBV-positive NK-cell and T-cell lymphoma; EBV-positive T-cell lymphoproliferation and childhood lymphoma, as well as all specific TCL entities within each of these subtypes according to the WHO classification.

[0316] Adult T-cell leukemia / lymphoma (ATL) is more common in Japan and the Caribbean than in the United States and is associated with human T-cell leukemia virus type 1 (HTLV-1), a rare and aggressive disease that begins in the liver or spleen and typically affects young adults in their 20s and 30s. Treatment for patients with hepatosplenic T-cell lymphoma includes anthracycline-based chemotherapy and, in some cases, stem cell transplantation.

[0317] Subcutaneous panniculitis-like lymphoma (SPTCL) is the rarest and least well-defined type of T-cell lymphoma. This lymphoma develops primarily in the fatty tissue beneath the skin, where it forms nodules. Symptoms include fever, chills, weight loss, and oral mucosal ulcers. SPTCL can be rapidly aggressive or indolent (slow-growing). Treatment includes chemotherapy with a combination of anthracyclines or localized radiation therapy. Precursor T-cell acute lymphoblastic lymphoma or leukemia may be diagnosed as leukemia, lymphoma, or both. This cancer is found in both children and adults, most often in adolescent and adult males. For patients newly diagnosed with precursor T-cell acute lymphoblastic lymphoma or leukemia, treatment is aggressive chemotherapy and radiation therapy. Nelarabine It is approved for the treatment of adults and children with relapsed or refractory T-cell precursor acute lymphoblastic lymphoma or leukemia.

[0318] Angioimmunoblastic T-cell lymphoma (AITL) is a tumor characterized by a strong inflammatory and immune response, as evidenced by its clinical, pathological, cellular, and biological characteristics. Because the tumor cells are phenotypically similar to T follicular helper (Tfh) cells, they are thought to function in some way similar to the non-neoplastic Tfh cells seen in reactive follicular hyperplasia. However, in the vast majority of AITL cases, the follicles do not proliferate but are exhausted or destroyed. Recently, it has been reported that AITL accounts for 36.1% of PTCL.

[0319] Cutaneous T-cell lymphoma (CTCL) accounts for about 70-75% of primary cutaneous lymphomas. CTCL may be a CTCL subtype selected from the group consisting of: mycosis fungoides (MF); Sezary syndrome (SS); and CD4+ small medium sized polymorphic T-cell lymphoproliferative disorder.

[0320] Mycosis fungoides (MF) is the most common subtype. Sézary syndrome (SS) is a more aggressive form of CTCL. Patients with SS have erythroderma (ie, rash affecting >80% body surface area [BSA]), lymphadenopathy, and high numbers of circulating tumor CD4+ T cells in the peripheral blood.

[0321] In other embodiments, the CAR constructs, effector cells, or pharmaceutical compositions can be used to treat any disease caused by pathogenic T cells.

[0322] In other embodiments, CAR constructs, effector cells or pharmaceutical compositions can be used to treat, prevent or improve autoimmune diseases. Autoimmune diseases can be caused by pathogenic self-reactive T cells, or are selected from the following: systemic lupus erythematosus, rheumatoid arthritis and myasthenia gravis.

[0323] It should be understood that the CAR constructs, nucleic acids, vectors, effector cells or pharmaceutical compositions of the present invention (collectively referred to herein as "agents") can be used for treatment in monotherapy (e.g., using CAR constructs, nucleic acids, vectors, effector cells or pharmaceutical compositions alone), preferably for (i) immunotherapy; (ii) for treating, preventing or improving cancer; (iii) for treating, preventing or improving autoimmune diseases; or (iv) for treating, preventing or improving any disease characterized by the presence of pathogenic T cells. Alternatively, the CAR constructs, effector cells or pharmaceutical compositions of the present invention can be used as an adjuvant to known immunotherapy or in combination with known immunotherapy, or for treating diseases caused by pathogenic T cells and cancer or autoimmune diseases.

[0324] The medicaments of the present invention can be combined into compositions having a variety of different forms, depending on how the composition is used. Thus, for example, the composition can be in liquid form, preferably delivered intravenously to a person in need of treatment. It should be understood that the pharmaceutical carrier of the present invention should be a carrier that is well tolerated by the subject receiving the treatment.

[0325] In a preferred embodiment, the agents and medicaments of the present invention can be administered to a subject by injection into the bloodstream or directly into the site in need of treatment. The injection can be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), intradermal (bolus or infusion), or directly into a tumor.

[0326] It should be understood that the amount of the required CAR construct, carrier or effector cell (i.e., agent) is determined by its biological activity and bioavailability, and bioavailability depends on the mode of administration, the physicochemical properties of the agent, and its use as a monotherapy or combination therapy. The frequency of administration is also affected by the persistence of the agent in the subject being treated. Those skilled in the art can determine the optimal dose to be administered, and will vary with the progress of the specific agent used, the strength of the pharmaceutical composition, the mode of administration, and the disease being treated (e.g., cancer, T cell malignancies, or autoimmune diseases). Other factors depending on the specific subject being treated (including the subject's age, weight, sex, diet, and administration time) will result in the need to adjust the dose.

[0327] In the tenth aspect, the present invention also provides a method for preparing the pharmaceutical composition of the sixth aspect, the method comprising combining a therapeutically effective amount of the CAR construct of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect or the effector cell of the fourth aspect with a pharmaceutically acceptable carrier.

[0328] A "subject" may be a vertebrate, a mammal, or a domestic animal. Most preferably, the subject is a human.

[0329] A "therapeutically effective amount" of a CAR construct, nucleic acid, vector, effector cell of a composition is any amount of the dose required to treat the disease being treated (e.g., cancer) or produce a desired effect when administered to a subject.

[0330] For example, a therapeutically effective amount of effector cells used may be at least 100, 1000 or 10,000 effector cells. Preferably, at least 100,000, at least 1,000,000 or at least 10,000,000 effector cells are used.

[0331] As used herein, "pharmaceutically acceptable carrier" is any known compound or combination of known compounds known to those skilled in the art that can be used to formulate a pharmaceutical composition. Preferably, in order to successfully achieve CAR effective therapy, a composition comprising CAR effector cells is prepared and then delivered as a cell suspension, most preferably intravenously.

[0332] The pharmaceutical carrier can be a liquid, and the pharmaceutical composition is in the form of a solution. The liquid carrier is used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent of the present invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat. The liquid carrier may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers or osmotic regulators. Examples of liquid carriers suitable for oral and parenteral administration include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols and polyols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used for compositions in the form of sterile liquids for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0333] Liquid pharmaceutical compositions are sterile solutions or suspensions, which can be used, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and particularly subcutaneous injection. The medicament can be prepared as a sterile solid composition, which can be dissolved or suspended in sterile water, saline or other suitable sterile injectable media when administered.

[0334] It should be understood that the present invention extends to any nucleic acid, peptide or variant, derivative or analog thereof, which substantially comprises an amino acid or nucleic acid sequence of any sequence as described herein, including variants or fragments thereof. The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" may be a sequence having at least 40% sequence identity with an amino acid / nucleotide / peptide sequence of any sequence as described herein, for example, 40% identity with a sequence identified herein, etc.

[0335] Also contemplated are amino acid / polynucleotide / polypeptide sequences having greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, still more preferably greater than 80% sequence identity with any sequence as described herein. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity with any sequence as described herein.

[0336] Those skilled in the art understand how to calculate the percent identity between two amino acid / polynucleotide / peptide sequences. In order to calculate the percent identity between two amino acid / polynucleotide / peptide sequences, the alignment of the two sequences must first be prepared, and then the sequence identity value is calculated. The percent identity of two sequences may take different values, depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (performed in different programs) or a structural alignment from a 3D alignment; and (ii) the parameters used by the alignment method, such as local alignment versus global alignment, the pairing score matrix used (e.g., BLOSUM62, PAM250, GonneT, etc.) and gap penalties, such as function forms and constants.

[0337] Once the alignment is complete, there are a number of different ways to calculate the percent identity between the two sequences. For example, the same number can be divided by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (v) the number of equivalent positions excluding overhangs. In addition, it should be understood that the percent identity also depends to a large extent on length. Therefore, the shorter a pair of sequences is, the higher the sequence identity that is expected to occur by chance.

[0338] Therefore, it should be understood that accurate alignment of protein or DNA sequences is a complex process. The commonly used multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882 pages) is a preferred method for generating protein or DNA multiple alignments of the present invention. Suitable parameters for ClustalW can be as follows: for DNA alignment: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, Matrix = Identity. For protein alignment: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, Matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, GAPDIST = 4. It is known to those skilled in the art that these and other parameters may need to be changed to achieve optimal sequence alignment.

[0339] Preferably, the percent identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated by an alignment such as (N / T)*100, where N is the number of positions of identical residues shared by the sequences and T is the total number of positions compared (including gaps and with or without overhangs). Preferably, the calculation includes overhangs. Thus, the most preferred method for calculating the percent identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a set of appropriate parameters (e.g., as described above); and (ii) substituting the values ​​of N and T into the following formula: sequence identity=(N / T)*100.

[0340] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by sequences that hybridize to a DNA sequence or its complementary sequence under stringent conditions. The inventors believe that stringent conditions refer to hybridization of nucleotides to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at about 20-65°C. Alternatively, substantially similar polypeptides may differ from the sequences as shown herein by at least 1 amino acid, but by less than 5, 10, 20, 50, or 100 amino acids.

[0341] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be modified or changed without significantly affecting the protein sequence encoded therein to provide a functional variant thereof. Suitable nucleotide variants are variants having sequences that are changed by replacing different codons encoding the same amino acid in the sequence, thereby producing silent (synonymous) changes. Other suitable variants are variants having homologous nucleotide sequences but comprising all or part of the sequence, which are changed by replacing different codons encoding amino acids with side chains having similar biophysical properties to the substituted amino acids, to produce conservative changes. For example, small non-polar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline and methionine. Large non-polar hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. Positively charged (alkaline) amino acids include lysine, arginine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood which amino acids can be substituted by amino acids having similar biophysical properties, and those skilled in the art know the nucleotide sequences encoding these amino acids.

[0342] All features described herein (including all accompanying claims, abstracts and drawings) and / or all steps of any method or process disclosed may be combined with any of the above aspects in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0343] For a better understanding of the present invention, reference is now made to the accompanying drawings, by way of example, to show how embodiments of the present invention may be implemented, in which:

[0344] Figure 1 A schematic diagram of an embodiment of the coding sequence of the anti-TCRVβCAR lentiviral CAR expression vector of the present invention is shown. The sequence includes a human CD8α signal peptide, an antigen binding domain comprising a VL and a flexible linker and a VH, a CD8α hinge domain comprising a transmembrane domain and a cytoplasmic domain, and an intracellular signaling domain comprising a CD28 costimulatory domain and a CD3ζ stimulatory domain.

[0345] Figure 2a The membrane topology of the mature CAR of the present invention is shown. The CD8α signal peptide, VL, linker and VH domains form an extracellular extracellular domain, and the CD8α hinge domain includes an extracellular domain portion, a plasma membrane (PM) and a cytoplasmic fragment. The CD28 costimulatory domain and the CD3ζ stimulatory domain form an intracellular intracellular domain; Figure 2bOne embodiment of the CAR of the present invention is shown, referred to as MPB2D5 CAR GS3 CD8283Z (TCRVB 2CAR).

[0346] Figure 3 The structural design details of the coding sequence of each of the multiple embodiments of the exemplary anti-TCRVβCAR of the present invention are provided. From top to bottom, the figure shows a CAR specific to TCRVβ1CAR (i.e., anti-TCRVβ1CAR), a CAR specific to TCRVβ2CAR (i.e., anti-TCRVβ2CAR), a CAR specific to TCRVβ9CAR (i.e., anti-TCRVβ9CAR), and a CAR specific to TCRVβ11CAR (i.e., anti-TCRVβ11CAR). For each embodiment, a CAR with 2x G 4 S linker constructs and those with 3x G 4 The position of each component of the construct (5' to 3') is based on Figure 1 A schematic diagram of the disclosed sequence is shown.

[0347] Figure 4 Shows Figure 3 The plasmid map of the anti-TCRVβ1CAR (called BL37.2 CAR) construct shown. The top image contains 2x G 4 The first embodiment of the anti-TCRVβ1CAR of the S linker, the bottom image is a CAR comprising 3x G 4 The second embodiment of the anti-TCRVβ1 CAR of the S linker.

[0348] Figure 5 Shows Figure 3 The plasmid map of the anti-TCRVβ2CAR (called MPB2D5 CAR) shown in Figure 1 is a plasmid containing 2x G 4 The first embodiment of the anti-TCRVβ2 CAR of the S linker, the bottom image is a CAR comprising 3x G 4 The second embodiment of the anti-TCRVβ2CAR of the S linker.

[0349] Figure 6 Shows Figure 3 The plasmid map of the anti-TCRVβ9CAR (called FIN9 CAR) shown in the figure. The top picture contains 2xG 4 The first embodiment of the anti-TCRVβ9 CAR of the S linker, the bottom image is a CAR comprising 3x G 4 The second embodiment of the anti-TCRVβ9CAR of the S linker.

[0350] Figure 7 Shows Figure 3The plasmid map of the anti-TCRVβ11CAR (called C21 CAR) shown in the figure. The top picture contains 2xG 4 The first embodiment of the anti-TCRVβ11 CAR of the S linker, the bottom figure is a 3x G 4 The second embodiment of the anti-TCRVβ11 CAR of the S linker.

[0351] Figure 8 The specificity, cytokine production, and cytotoxic activity of the anti-TCRVβCAR-T cells of the present invention were shown. Peripheral blood mononuclear cells (PBMCs) from normal donors were cultured alone or transduced with lentivirus encoding anti-Vβ1, Vβ2, and Vβ9CAR constructs. Five days after transduction, transduction efficiency was assessed by protein L staining (see Figure 8 A and 8B ), and quantified the frequency of CD3+ cells expressing each of the 24 TCRVβ subunits (see Figure 8 C). The values ​​shown are the mean percentage of cells expressing each subunit, which was normalized to the frequency of cells expressing the subunit in non-transduced PBMC controls from two (n=2) individuals. Anti-TCR CAR-T cells and anti-CD19 CAR-T cells killed in vitro expanded autologous primary T cell lines (see Figure 8 D), anti-VβCAR-T cells killed untransduced (UT) and TCR-GFP transduced (Vβ+) JRT3-T3.5 cells (see Figure 8 E). Target cells were stained with CFSE and co-cultured with effector cells at a range of effector to target (E:T) ratios in duplicate. After 24 h, cells were harvested and stained with 7aad. The frequency of dead target cells in each culture condition was determined by flow cytometry. Results are representative of more than two independent experiments. CD107a mobilization, IFN-γ and TNF-α production of CD3+ anti-Vβ2 and anti-CD19 CAR-T cells cultured alone or in the presence of untransduced JRT3-T3.5 cells (JRT), JRT3-T3.5 cells transduced with Vβ2+TCR-GFP (TCR GFP) or primary cells expressing Vβ2 (1YT) are shown (see Figure 8 F) Results are from a single experiment and are representative of two independent experiments.

[0352] Fig. 9 The cytokine production and cytotoxic activity of the anti-TCRVβCAR-iNKT cells of the present invention are shown. Anti-TCRCAR-iNKT cells and anti-CD19 CAR-iNKT kill autologous primary T cell lines expanded in vitro (see Fig. 9A). Anti-VβCAR-iNKT cells kill untransduced (UT) and TCR-GFP-transduced (TCR-GFP) JRT-3T3.5 cells (see Fig. 9 B). Target cells were stained with CFSE and co-cultured with effector cells at a range of effector cell to target cell (E:T) ratios, repeated twice. After 24 hours, cells were collected and stained with 7AAD. The frequency of dead target cells in each culture condition was determined by flow cytometry. Results are representative of more than two independent experiments. CD107a mobilization, IFN-γ and TNF-α production by CD3+ anti-Vβ2 and anti-CD19 CAR-T cells cultured alone or in the presence of untransduced JRT3-T3.5 cells (JRT), JRT3-T3.5 cells transduced with Vβ2+TCR-GFP (Vβ+), or primary cells expressing Vβ2 (1YT) are shown (see Fig. 9 C). Results are from a single experiment and are representative of 2 independent experiments. Anti-Vβ1CAR-iNKT (see Fig. 9 D) or anti-Vβ2CAR-iNKT, but not anti-Vβ1CAR-T cells or anti-Vβ2CAR-T cells (see Fig. 9 E), enhanced killing of untransduced and TCRVβ1 or TCRVβ2-GFP transduced JRT3-T3.5 cells loaded with α-gal. As shown in the results, target cells were incubated with 200 ng / ml α-gal before co-culture with effector cells. The results shown are representative of two replicate experiments. The in vivo protocol used was as Fig. 9 As shown in F. 5x10 6 TCRVβ+JRT cells were suspended in matrigel and injected subcutaneously. 6 Effector cells were injected intravenously into the tail vein. The challenged groups consisted of an untreated group (n=5), a Vβ2CAR-iNKT group (n=7), or a CD19CAR-iNKT group (n=5). The tumor volume of each group was measured regularly using a caliper (see Fig. 9 G). At the end of the experiment, the tumors were removed and weighed (see Fig. 9 H).

[0353] Fig.10 The anti-VβCAR-iNKT cells of the present invention kill adult T cell leukemia / lymphoma (ATL) cells in vitro. Patient ATL01 has a malignant clone expressing TCRVβ1, and patient ATL02 has a malignant clone expressing TCRVβ2 (see Fig.10 A). Anti-VβCAR-iNKT cells kill TCRVβ1 in vitro (see Fig.10 B) and TCRVβ2 (see Fig.10C) CD4+CCR4+CD26-ATL cells. Cryopreserved PBMCs from two ATL patients and a normal donor (ND) were stained with Cell-TraceViolet and co-incubated with anti-Vβ1CAR-iNKT, anti-Vβ2CAR-iNKT, or anti-CD19 CAR-iNKT at the indicated effector cell:target cell (PBMC) ratios in triplicate. After 24 hours of co-culture, cells were stained with viability dyes, anti-CD4, CCR4, CD26, TCRVβ1 or TCRVβ2, and TCRαβ and fixed, and the frequency of dead CD4+CCR4+CD26- T cells and CCR4-CD26+ / - cells (“remaining” CD4+ T cells) was determined by flow cytometry. *The number of CD4+ cells expressing other TCRVβ subunits in patient 2 was insufficient to determine their viability.

[0354] Fig.11 The effect of CAR-iNKT cells on the frequency of antiviral CTLs and CD4+T cells expressing HTLV-1 is shown. The frequency of M158-66 and Tax11-19 HLA-A*0201 pentamer+CD3+T cells after co-culture with CAR-iNKT is shown (see Fig.11 A and 11B). CD4-depleted PBMCs from three HLA-A*0201+HTLV-1 carriers were stained with CellTrace Violet and cultured alone or with anti-Vβ1CAR-iNKT, anti-Vβ2CAR-iNKT, or CD19 CAR-iNKT at a 1:1 ratio. After 16-18 h of co-culture, cells were stained with viability dye and anti-CD3, anti-CD8, and M1 58-66 or Tax 11-19 Cells were stained with Pentamer. Cells were analyzed by flow cytometry and the frequency of viable Pentamer+CellTraceViolet+CD3+ cells was determined. Fig.11 C and 11D show the frequency of CD4+ cells expressing HTLV-1Tax after co-culture with CAR-iNKT (see Fig.11 C and 11D). CellTrace Violet-stained positively selected CD4+ cells from the same donor were cultured alone or in the presence of Vβ2-CAR iNKT or CD19-CAR iNKT. After 16-18 hours, the cells were stained with viability dye, anti-CD3, anti-CD4, anti-CD8, and anti-TCRVβ2. The cells were then fixed and intracellularly stained with anti-Tax antibody. The cells were analyzed by flow cytometry, and the frequency of TCRVβ2+CD3+ live cells and Tax+CD4+ live cells was determined.

[0355] Fig.12 Shows Figure 8 Raw data for c: i.e., absolute frequency of CD3+ T cells expressing each TCRVβ subunit, expressed as a percentage of total CD3+ T cells.

[0356] Fig.13 The target cell lines of the CAR T cells of the present invention are shown. Anti-TCRVβ subunit antibodies and magnetic beads are used to enrich primary T cells expressing the Vβ subunit of interest (see Fig.13 A). JRT3-T3.5 cells were transduced with lentiviral expression vectors encoding TCRβ chain and GFP (see Fig.13 B). Cells were sorted to enrich for cells expressing CD3 on the cell surface. One week after sorting, cells were stained with antibodies specific for CD3 and the associated TCRVβ subunit and analyzed by flow cytometry.

[0357] Fig.14 The iNKT iTCR cells of the present invention play a role in killing T cells. Primary T cells expanded in vitro and JRT3-T3.5 cells (JRT) express CD1d (see Fig.14 A). Cells were stained with viability dye, anti-CD3 and anti-CD1d and analyzed by flow cytometry. CD3 and CD1d expression of viable cells is shown. Fig. 9 F-9H shows the body weight of mice during the in vivo experiment. (See Fig.14 B).

[0358] Fig.15 The effect of the CAR-iNKT cells of the present invention on CD4+T cells expressing HTLV-1 is shown. The frequency of CD4+ cells expressing HTLV-1Tax after co-culture with CAR-iNKT is shown (see Fig.15 A and 15B). Fig.15 C shows the frequency of TCRVβ1 and TCRVβ2+CD3+T cells after co-culture. CellTrace Violet staining was performed, and positively selected CD4+ cells from the same donor were cultured alone or in the presence of Vβ1-CAR iNKT or CD19-CAR iNKT. After 16-18 hours, the cells were stained with viability dye, anti-CD3, anti-CD4, anti-CD8, and anti-TCRVβ1. The cells were then fixed and intracellularly stained with anti-Tax antibody. The cells were then analyzed by flow cytometry, and the frequency of TCRVβ1+ or TCRVβ2+CD3+ live cells and Tax+CD4+ live cells was determined.

[0359] Fig.16A comparison of the effect of linker size on CAR-T efficacy is provided. Anti-TCR CAR-T cells and anti-CD19 CAR-T kill autologous primary T cell lines expanded in vitro. Target cells were stained with CFSE and co-cultured with effector cells at a range of effector cell to target cell (E:T) ratios, repeated twice. After 24 hours, cells were collected and stained with 7aad. The frequency of dead target cells under each culture condition was determined by flow cytometry. DETAILED DESCRIPTION

[0360] Example

[0361] As described below, the inventors generated exemplary anti-TCRVβCAR constructs (herein, for TCR Vβ1, referred to as clone BL37.2; for TCR Vβ2, referred to as clone MPB2D5; for TCR Vβ9, referred to as clone FIN9; for TCR Vβ11, referred to as clone C21) (see Example 1), and tested their activity against primary T cells and cancer T cell lines that specifically express selected TCRVβ (see Example 2). The inventors then generated effector T cells expressing selected anti-TCRVβCAR constructs, in particular iNKT cells expressing selected anti-TCRVβCAR constructs, and analyzed their suitability for immunotherapy of T cell malignancies (see Example 3). The inventors then successfully demonstrated that anti-TCRVβCAR-iNKT cells are highly active against ATL (see Example 4), and do not impair CTL immunity against viral antigens, nor promote the replication activity of HTLV-1, thereby exhibiting important safety features required for the treatment of ATL and TCL.

[0362] Materials and methods

[0363] Ethical Statement

[0364] Patients with ATL presented to the National Centre for Human Retrovirology (Imperial College Healthcare NHS Trust, St Mary's Hospital, London), and written informed consent was obtained. Studies involving samples from patients with ATL were conducted under the care of the Infectious Diseases Research Group Tissue Bank and were approved by the United Kingdom National Research Ethics Service (09 / H0606 / 106, 15 / SC / 0089, 20 / SC / 0226).

[0365] Cell lines and chemicals

[0366] HEK 293T cells were passaged twice a week by trypsinization and maintained at 30–70% confluence in complete Dulbecco's modified eagle medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine. JRT3-T3.5 cells were obtained from ATCC and maintained at 1×10 cells / mL in complete RPMI medium or iNKT medium. 5 -1x10 6 C1R-CD1d cells (gift from Vincenzo Cerundolo, University of Oxford) were maintained at 1×10 cells / ml in complete RPMI medium containing 10% (v / v) FBS, 100 units / ml penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine, and iNKT medium (RPMI containing 10% FBS, 15 mM Hepes, 1 mM sodium pyruvate, 1× MEM non-essential amino acids, 4 mM L-glutamine, 0.05 mM β-mercaptoethanol, 100 units / ml penicillin, and 100 μg / mL streptomycin). 5 -1x10 6 cells / ml.

[0367] Generation of CAR constructs

[0368] Four monoclonal antibodies were identified for proof-of-principle studies, directed against unbound TCR Vβ1 (clone BL37.2), TCR Vβ2 (MPB2D5), TCR Vβ9 (FIN9), and TCR Vβ11 (C21) purchased from Beckman Coulter. The amino acid sequences of the heavy and light chain variable regions of BL37.2, MPB2D5, and FIN9 were determined by mass spectrometry (Rapid Novor, Canada). For clone C21, total RNA was extracted from hybridoma cells, reverse transcribed, and isotype-specific primers were used to amplify the heavy and light chain fragments. The amplified fragments were cloned into a standard cloning vector, transformed into Escherichia coli, and five colonies for each region of interest were sequenced by Sanger sequencing (GenScript). Codon-optimized gene fragments were synthesized by Genewiz and cloned into lentiviral expression vectors to generate Figure 1 and Fig.12 Constructs as indicated.

[0369] Lentivirus production

[0370] HEK293T cells were cultured at 4 x 10 6The density of each cell / 10cm plate was inoculated in 10ml complete DMEM. After 24 hours, the lentiviral expression vector and the second generation packaging plasmid were transfected into the cells using GeneJuice (from Merck). 48 hours after transfection, the supernatant was collected, clarified by centrifugation at 500g for 5 minutes, and then passed through a 0.45μM filter. The lentiviral particles were concentrated by ultracentrifugation at 23,000rpm for 2 hours at 4°C, resuspended in 200-300μl serum-free RPMI, and stored at -80°C until use. The virus titer was determined by titration of HEK-293T cells.

[0371] Isolation of peripheral blood mononuclear cells

[0372] Peripheral blood mononuclear cells (PBMC) were purified from apheresis cones or EDTA anticoagulated blood by density gradient centrifugation. Briefly, cells were collected from the cones and diluted to 100 ml in PBS. 25 ml of the diluted cells were layered on 15 ml of histopaque (Sigma) and centrifuged at 800 g for 20 minutes. Buffy coat cells were collected, washed with PBS (400 g, 5 minutes at room temperature), and stored frozen in FBS containing 10% dimethyl sulfoxide (DMSO) until use.

[0373] Lentiviral transduction of T cells and iNKT cells

[0374] PBMCs were thawed quickly, washed twice in RPMI containing 10% FBS, and resuspended in a small amount of culture medium. According to the manufacturer's instructions (Miltenyi Biotech), magnetic bead separation was used to positively select iNKT, and the cells were passed through two consecutive LS columns. The purity of the positive part was assessed by flow cytometry staining using a fixable viability dye (Live / Dead near infrared, Life Sciences) and anti-TCRVα24-Jα18BV421 (clone 6B11), anti-TCRVΒ11APC, anti-CD3 BV510, anti-CD4 BV605, and anti-CD8 FITC. For iNKT culture, the positively selected part was cultured with irradiated (35Gy) autologous feeder cells prepared from the negative part at a ratio of 1: 1. For T cell culture, the flow-through part of iNKT depletion was cultured alone. T cells and iNKT cells were cultured in iNKT medium and stimulated with 50 ng / ml anti-CD3, 50 ng / ml anti-CD28 (Miltenyi-Biotech), and 150 IU / ml IL-15. After 48 h of culture, the required volume of each lentivirus was placed in fibronectin-coated plates to give a multiplicity of infection (MOI) of 2.5-5 infectious units per cell. Cells were collected, counted, and added to the transduction plate, followed by centrifugation at 1000 g for 40 min at 32 °C. On day 7 of iNKT culture, feeder cells expressing CD1d were added with 200 ng / ml α-galactosylceramide (αGalCer, BioVision) for 2-4 h and cultured at a 1:1 ratio after irradiation. Cells were fed with fresh medium containing 150 IU / ml IL-15 twice a week.

[0375] Quantification of lentiviral transduction efficiency

[0376] 5 days after transduction, start to evaluate transduction efficiency. Wash cells twice in cold PBS and incubate with 1 μg / ml streptavidin protein L (Pierce) at 4°C for 45 minutes. Wash cells twice with PBS, stain with Live / Dead near infrared for 5 minutes at room temperature, wash with PBS 0.5% FBS, and stain with streptavidin-PE / BV421 and anti-CD3, anti-CD4, anti-CD8, anti-TCRVβ11 and anti-TCRVα24-Jα18 for 20 minutes at room temperature. On the same day, cells were obtained using BD Fortessa and analyzed using Kaluza (Beckman Coulter).

[0377] Isolation and culture of primary T cell targets

[0378] Autologous primary T cell lines expressing the investigated TCRVβ subunit were established by magnetic enrichment and in vitro expansion. PBMCs were stained with PE-conjugated anti-TCRVβ antibody (Beckman Coulter) at room temperature for 20 minutes, washed once in complete RPMI, and then incubated with anti-PE microbeads (Miltenyi Biotech) at 4°C for another 20 minutes. After washing once more, the cells were passed through two consecutive LS columns. The positive fraction was cultured with anti-CD3, anti-CD28, and 150 IU / ml IL-2.

[0379] Generation of GFP-TCR (cells expressing JRT3-T3.5)

[0380] TCRβ chain sequences containing the Vβ subunit of interest were identified in public databases. The following table summarizes the nomenclature of TCR genes (TRBV) and proteins (TCRVβ) and indicates the proteins to which commercially available monoclonal antibodies are derived. Codon-optimized nucleotide sequences corresponding to the full-length β chain were synthesized (using Genewiz) and cloned into a third-generation lentiviral expression vector (LeGO-iG2).

[0381] Table 2: TCR gene (TRBV) and protein (TCRVβ) nomenclature, including proteins to which commercially available monoclonal antibodies are available.

[0382]

[0383]

[0384] 7AAD cytotoxicity assay

[0385] The day before assessing cytotoxicity, effector cells were counted and fed with fresh medium. Target cells were washed twice with PBS, incubated in 0.5 μM CFSE / CellTrace Violet for 10 minutes at 37°C, washed twice in iNKT medium, and then returned to culture (with or without αGalCer as needed). On the day of the experiment, cells were centrifuged and resuspended in iNKT medium containing 15 IU / ml IL-15. Cells were mixed to achieve a range of effector cell: target cell (E:T) ratios, placed in U-bottom 96-well plates (repeated twice), and centrifuged at 100g for 1 minute. Cells were cultured for 4-24 hours, centrifuged at 800g for 3 minutes, and resuspended in PBS containing 7aad (5μg / ml). After incubation for 20 minutes, each well was washed with 150μl PBS, centrifuged at 800g for 3 minutes, and then resuspended with 100μl PBS. In each well, the frequency of dead (7AAD+) target cells was counted by flow cytometry.

[0386] Fresh PBMC cytotoxicity assay

[0387] The day before evaluating cytotoxicity, effector cells were counted and fed with fresh medium. On the day of the experiment, cryopreserved PBMCs were thawed, washed once with iNKT medium (400g, 5 minutes), washed once with PBS, incubated in 0.5μM CFSE / CellTrace Violet at 37°C for 10 minutes, and washed twice in iNKT medium. PBMCs were placed in a U-bottom 96-well plate containing 15IU / ml IL-15 (with or without CAR-iNKT cells) with a range of effector cells: target cell ratios, repeated three times. The plate was centrifuged at 100g for 1 minute and cultured for 24 hours. The wells were washed with 150 μl PBS (800 g, 3 minutes), stained with Live / Dead near infrared for 5 minutes at room temperature, washed with FACS buffer (PBS 7% (v / v) NGS), and stained with anti-TCRVβ-PE, anti-TCRαβ-FITC, anti-CD4-BV605, anti-CCR4-APC and anti-CD26-PeCy7 in FACS buffer at room temperature for 20 minutes. The cells were washed with 150 μl FACS buffer, fixed with 150 μl fixed buffer (Biolegend) for 20 minutes, washed once with FACS buffer, and then stored at 4°C until collection.

[0388] Intracellular cytokine staining and degranulation assay

[0389] Twenty-four hours (24h) before evaluating intracellular cytokine production / degranulation, effector cells were fed and target cells were stained with CFSE. On the day of the experiment, target cells and effector cells were collected, counted and mixed, and a 1:1 E:T ratio was obtained in iNKT culture medium containing 20μg / ml deoxyribonuclease (DNase), 15IU / ml IL-15, 10.6μM brefeldin, 2μM monensin (1x protein transport inhibitor, eBioscience) and 2.5μl anti-CD107a-BV421 (clone H4A3) / 200μl culture wells. Cells were cultured for 6 hours, centrifuged at 800g for 3 minutes to remove the culture supernatant, and resuspended in Live / Dead NIR vitality dye. After incubation at room temperature for 5 minutes, 150μl PBS 0.5% FBS was added to each well and the plate was washed again. The cells were resuspended in 150 μl of fixation / permeabilization buffer (eBiosciences FoxP3 buffer set, Life Technologies), incubated for 30 minutes at room temperature, and washed once in permeabilization buffer. The cells were stained with anti-CD3-BV510 (clone UCHT-1)-CD4-BV605 (RPA-T4)-CD8-AF700 (RPA-T8)-IFN-γ-BV711 (4S.B3), anti-TNF-α-PeCy7 (Mab11), granzyme B-PE (QA16A02) and perforin-APC (B-D48) diluted in permeabilization buffer. After incubation for 30 minutes at room temperature, the cells were washed once with 150 μl of permeabilization buffer and resuspended with PBS 0.5% FBS until collection.

[0390] Pentamer staining

[0391] HTLV-1 infected CD4+ cells express viral antigens in short-term in vitro culture and can therefore present Tax11-19 peptides in the presence of HLA-A*0201. Therefore, to minimize the possibility of downregulation of cognate TCRs on CTLs due to antigen encounter during culture, CD4+ T cells were depleted from PBMCs of HLA-A*0201+HTLV-1 carriers using anti-CD4 PE and anti-PE microbeads as described above. CD4-depleted PBMCs were stained with Cell Trace Violet. Positive and negative fractions were cultured in iNKT medium containing 15 IU / ml IL-15 in the presence or absence of CAR-iNKT cells. After 16-18 hours of co-culture, the cells were stained with Live / Dead near infrared as described above and resuspended with 10 μl HTLV-1Tax11-19 or influenza A M158-66 pentamer-APC in 40 μl PBS for 10 minutes, and then anti-CD3-BV510 and anti-CD8-AF700 were added. After incubation at room temperature for another 20 minutes, the cells were washed and fixed for 30 minutes by suspending the cells in 150 μl fixation buffer (Biolegend). After washing once with PBS, the frequency of live pentamer-positive cells Cell Trace Violet+CD8+T cells in each culture was assessed by flow cytometry. Similarly, the CD4+ portion was stained with Live / Dead, anti-CD4-BV605, and anti-CD3-BV510, washed with PBS 7% NGS, and fixed with 150 μl ebioscience FoxP3 fixation / permeabilization buffer for 30 minutes. The cells were then washed once in ebioscience permeabilization buffer and stained intracellularly with anti-TaxAF647 for 30 minutes. The cells were washed once more with 150 μl permeabilization buffer, resuspended in PBS, and stored at 4°C in the dark until harvesting. The number of Tax+CD4+CellTrace Violet+ cells was measured by flow cytometry.

[0392] In vivo experiments

[0393] 5x10 6 TCRVβ+JRT cells were suspended in Matrigel and injected subcutaneously into the flank of NSG mice. 6 Effector cells were injected intravenously into the tail vein. Groups consisted of an untreated group (n=5), a Vβ2CAR-iNKT group (n=7), or a CD19CAR-iNKT group (n=5). Tumor volume in each group was measured regularly using a caliper. At the end of the experiment, the tumor was removed and weighed.

[0394] Off-target killing assay

[0395] PBMCs from two normal donors were cultured alone or transduced with lentivirus encoding Vβ1, Vβ2, Vβ9, and Vβ11 specific CAR constructs. Five days after transduction, transduction efficiency was assessed by protein L staining, and the frequency of CD3+ cells expressing each of the 24 TCRVβ subunits was quantified. The reported value is the average percentage of cells expressing each subunit, which is normalized to the frequency of cells expressing the subunit in the untransduced PBMC control from each individual.

[0396] result

[0397] Example 1: Generation of CAR constructs targeting TCRVβ1, TCRVβ2, TCRVβ9, and TCRVβ11

[0398] The inventors generated four lentiviral CAR constructs to target TCRVβ1 (expressed on 3.5% CD3+ T cells of healthy donors), TCRVβ2 (8.3%), TCRVβ9 (3.2%), and TCRVβ11 (1%). The anti-TCRVβ1 CAR is called "BL37.2CAR", the anti-TCRVβ2 CAR is called "MPB2D5 CAR", the anti-TCRVβ9 CAR is called "FIN9CAR", and the anti-TCRVβ11 CAR is called "C21 CAR".

[0399] Since hybridoma clones could not be obtained for the first three monoclonal antibodies, the inventors reverse engineered the CAR after determining the amino acid sequence of the monoclonal antibodies by mass spectrometry. The corresponding nucleotide sequence was codon-optimized for expression in human cells and used to construct a second-generation CAR in which the external domain contained the CD8α leader peptide -VL-(GGGGS) 2 The linker-VH-CD8α hinge-CD8α transmembrane domain, while the intracellular domain consists of a portion of the CD8α cytoplasmic domain and the signaling (i.e., stimulatory and co-stimulatory) domains of CD28-CD3ζ (see Figure 1 and 2).

[0400] refer to Figure 1 2, a schematic diagram of an embodiment of the coding sequence of the expression vector of the anti-TCRVβCAR lentiviral CAR of the present invention is shown. It should be understood that the vector (DNA) also corresponds to CAR (protein). The CAR comprises a human CD8α signal peptide, an antigen binding domain comprising VL and a flexible linker and VH, a CD8α hinge domain comprising a transmembrane domain and a cytoplasmic domain, and an intracellular signaling domain comprising a CD28 costimulatory domain and a CD3ζ stimulatory domain.

[0401] To generate an anti-TCRVβ11 CAR with codon-optimized VH / VL sequences, the inventors amplified the expressed VH and VL chains using mRNA extracted from hybridoma C21. The resulting CAR construct was Figure 3 For each of the four types of CAR (i.e., “BL37.2 CAR,” “MPB2D5 CAR,” “FIN9 CAR,” and “C21 CAR”), a 2x G 4 S linker constructs and those with 3x G 4 The position of each component of the construct (5' to 3') is based on Figure 1 A schematic diagram of the disclosed sequence is shown.

[0402] The signal peptide is the same as that of human CD8α. Because the signal peptide matches the type of effector cells (as described below), and CD8α is highly expressed in T cells, the inventors believe that their signal peptide will provide the optimal expression of CAR constructs in effector cells. The hinge region of CAR includes amino acids 128-210 of human CD8α, including 7 amino acids from 55 amino acids, a full transmembrane helical domain, and a cytoplasmic domain from an extracellular domain. The intracellular domain of CAR includes a CD3ζ stimulatory domain and a CD28 costimulatory domain.

[0403] The plasmids encoding the lentiviral CAR constructs targeting TCRVβ1, TCRVβ2, TCRVβ9, and TCRVβ11 were respectively as described in Figure 4 , 5 , 6 and 7 as shown.

[0404] Example 2: Evaluation of the specificity, cytokine production, and cellularity of effector cells expressing anti-TCRVβ CAR constructs Toxic activity

[0405] To begin testing the activity and specificity of the CARs described in Example 1, the inventors transduced them into PBMC T cells from two healthy donors by lentiviral transduction. Five days after transduction, CAR expression on T cells exceeded 50% ( Figure 8 A and 8B). At the same time, with reference to untransduced T cells, the inventors quantified the frequency of T cells expressing each of the 24 TCRVβ chains in CAR-transduced PBMCs. The inventors found that T cells expressing TCRVβ1, TCRVβ2, TCRVβ9, and TCRVβ11 were almost completely depleted in PBMCs transduced with the corresponding homologous CAR constructs (reduction of 98% (median); range 90-100%) ( Figure 8 C and Fig.12). In contrast, the inventors observed that the median frequency of non-targeted Vβ subunits in transduced cultures increased by 4% relative to non-transduced controls, indicating a lack of “off-target” killing by TCR Vβ family-specific CAR-T cells.

[0406] In summary, anti-TCRVβ1CAR, anti-TCRVβ2CAR, anti-TCRVβ9CAR, and anti-TCRVβ11CAR expressed robustly, were active, and were selective for their cognate TCRVβ chain targets.

[0407] Example 3: Anti-TCRVβ chain CAR for antigen generation and cancer T cell lines

[0408] To further investigate the anti-T cell activity and TCRVβ specificity of the four CAR constructs, the inventors first tested the effects of TCRVβCAR-T cells on an expanded autologous primary T cell line that was highly purified to express the TCRVβ chain of interest ( Fig.13 A). The inventors found that all four anti-TCRVβCAR-T cells selectively killed their homologous T cell lines. However, the levels of cytotoxicity were different and corresponded to the staining intensity of the corresponding monoclonal antibodies, with anti-TCRVβ1 and TCRVβ2 having the highest cytotoxicity and TCRVβ9 having the lowest cytotoxicity ( Figure 8 D).

[0409] Next, the inventors engineered the JRT3-T3.5T cell line to express either the TCRVβ1 or TCRVβ2 chain as a target. JRT3-T3.5 is a derivative of the Jurkat T cell line, which itself is derived from a patient with TCRVβ+ lymphoblastic T-cell lymphoma. Due to the loss of the endogenous TCRVβ chain gene, JRT3-T3.5 cells lack expression of TCR and CD3, so, as expected, the introduction of exogenous TCRVβ cDNA can restore the expression of both ( Fig.13 B). Therefore, the inventors found that anti-TCRVβ1 and two CAR-T cells kill JRT3-T3.5 cells expressing their homologous target TCRVβ, but not the parental cell line ( Figure 8 E).

[0410] In a complementary functional approach, the inventors found that, upon intracellular staining, anti-TCRVβ2 CAR-T cells expressed IFNg, TNFa, and CD107a (indicating cytotoxic degranulation) when co-cultured with JRT3-T3.5 cells or primary T cell lines expressing TCRVβ2, but not in the presence of parental JRT3-T3.5 or when cultured alone, and that more than 30% of CD4+ and CD8+ CAR-T cells were polyfunctional, i.e., co-expressed at least two molecules ( Figure 8E and 8F).

[0411] In summary, these findings demonstrate that the anti-TCRVβ CAR of the present invention developed by reverse engineering is highly specific and active in vitro.

[0412] Example 4: Anti-TCRVβCAR-iNKT cells for "off-the-shelf" immunotherapy of T cell malignancies

[0413] Early studies have demonstrated the feasibility of using iNKT cells as effector cells for CAR immunotherapy of blood cancer. Therefore, the inventors used their own preparation scheme to generate anti-TCRVβ1, anti-TCRVβ2, anti-TCRVβ9 and anti-TCRVβ11 CAR-iNKT cells. Anti-TCRVβ1, anti-TCRVβ2 and anti-TCRVβ9 CAR-iNKT cells effectively killed JRT3-T3.5 and primary T cell lines expressing the corresponding TCRβ chains ( Fig. 9 A and 9B), while CD4+ and CD4-CAR-iNKT cells specifically expressed or co-expressed IFNg, TNFa, and CD107a after stimulation with JRT3-T3.5 and primary T cells expressing TCRVβ2 ( Fig. 9 C).

[0414] Since JRT3-T3.5 cells express CD1d ( Fig.14 A), the inventors tested whether the responsiveness of anti-TCRVβCAR-iNKT cells could be enhanced in the presence of aGC. aGC is a glycolipid ligand for CD1d that can potently and selectively activate iNKT cells 16,17 The inventors found that although the cytotoxicity of anti-TCRVβ1CAR-T and anti-TCRVβ2CAR-T to parental JRT3-T3.5 cells homologously expressing TCRVβ was not enhanced in the presence of aGC, the cytotoxicity of anti-TCRVβ1CAR-iNKT and anti-TCRVβ2CAR-iNKT to both targets was enhanced, and the cytotoxicity to parental JRT3-T3.5T cells was greater ( Fig. 9 D and 9E), thus demonstrating the functional relevance of iTCR in the anti-tumor activity of CAR-iNKT cells.

[0415] The inventors further tested the activity of TCRVβCAR-iNKT cells in an in vivo T-cell lymphoma model, in which JRT3-T3.5 T cells expressing TCRVβ2 were injected subcutaneously into the flank of NSG mice ( Fig. 9 F) After tumor implantation, mice received either no treatment or 10 6In contrast, TCRVβ2CAR-iNKT cells significantly inhibited tumor volume and weight ( Fig. 9 G and Fig. 9 H).

[0416] Finally, mice treated with CAR-iNKT cells showed no evidence of excessive weight loss or other clinical signs of aGVHD after 42 days of monitoring ( Fig.14 B), thus supporting the idea of ​​using allogeneic iNKT cells as an “off-the-shelf” platform for cancer immunotherapy without the risk of aGVHD.

[0417] These data collectively demonstrate the therapeutic potential of the allogeneic TCRVβCAR-iNKT cells of the present invention for the treatment of T-cell lymphoma.

[0418] Example 5: Anti-TCRVβCAR-iNKT cells are active against ATL

[0419] Similar to the tests performed on JRT3-T3.5 T cell lymphoma cells, the inventors also tested the reactivity of allogeneic anti-TCRVβ1 CAR-iNKT and anti-TCRVβ2 CAR-iNKT cells against PBMCs from normal donors and PBMCs from two ATL patients with lymphoma cells clonally enriched for TCRVβ1 or TCRVβ2 expression ( Fig.10 A). In addition to clonal TCRs, ATL malignant cells also co-express CD4 and CCR4, and are negative for CD26, as previously demonstrated by the inventors ( Fig.10 A). Therefore, the inventors co-cultured matched anti-TCRβ CAR-iNKT and anti-CD19 CAR-iNKT with PBMC from each ATL and normal donor at a range of E:T ratios. Since co-culture with anti-TCRVβ CAR-iNKT cells prevented subsequent staining with the same anti-TCRVβ antibody clone, as previously reported by the inventors, the inventors used the immunophenotype CD4+CCR4 中 / 高 The inventors found that in patients 1 and 2, CCR4+CD26- cells accounted for 90% of CD4+ cells, and almost all expressed TCRVβ1 or TCRVβ2, respectively, while in normal donors, CCR4+CD26- cells accounted for 25% of CD4+ cells, and TCRVβ1 and TCRVβ2 accounted for less than 8%.

[0420] When the effector cell:CD4+T cell ratio was 1:1, anti-TCRVβ1CAR-iNKT killed 75% of CD4+CCR4+CD26- cells in patient 1, as well as 10% of "other" CD4+ cells (i.e., CCR4-CD26-). When the ratio was the same in patient 2, anti-TCRVβ2CAR-iNKT killed 54% of CD4+CCR4+CD26- cells. The frequency of CD4+ cells expressing other TCRVβ subunits was too low to determine whether there was no off-target killing of normal CD4+ cells in this patient. Minimal killing of CD4+CCR4+CD26- cells and "other CD4+" cells was observed when PBMCs from patients or normal donors were co-cultured with anti-CD19 CAR-iNKT cells. Similarly, minimal killing was observed after co-culture of PBMCs from normal donors with anti-TCRVβ1CAR-iNKT and anti-TCRVβ2CAR-iNKT cells, likely due to the lower frequency of target cells present in the analyzed populations ( Fig.10 B and 10C).

[0421] In conclusion, anti-TCRVβCAR-iNKT cells were highly active and specific against primary ATL cancer cells.

[0422] Example 6: Effects of anti-VβCAR-iNKT on antiviral CTL immunity and HTLV-1 viral status

[0423] To further demonstrate selective targeting, the inventors studied the in vitro effects of the anti-TCRVβCAR-iNKT cells of the present invention on antiviral CD8+T cells in HTVL-1 infected individuals. To this end, the inventors cultured PBMCs from three HLA-A*0201+HTLV-1 infected individuals (i.e., carriers) alone or in the presence of CAR-iNKT cells and evaluated the frequency of T cells bound to HLA-A*0201HTLV-1Tax11-19 or influenza A AM158-66 peptide-MHC pentamers ( Fig.11 A and 11B). When cultured alone, the frequency of influenza A M158-66 pentamer + CD3 + T cells ranged from 0.14-.23% of CD3 + cells, and the frequency of HTLV-1 Tax11-19 pentamer + CD3 + T cells ranged from 0.23-0.88%. When co-cultured with Vβ1-CAR-iNKT, Vβ2-CAR-iNKT, or CD19-CAR-iNKT cells, the frequency of pentamer + cells did not change ( Fig.11 A and 11B). In parallel cultures from the same donor, it was confirmed that homologous CAR-iNKT cells effectively depleted T cells expressing TCRVβ1 and TCRVβ2 ( Fig.15 C).

[0424] Since CAR-iNKT cell killing of target cells is associated with the secretion of inflammatory cytokines, the inventors wondered whether CAR-iNKT killing of target cells would enhance the spontaneous expression of HTLV-1Tax, a process that could promote HTLV-1 infection of uninfected CD4+T cells. To this end, positively selected CD4+T cells from the same three HTLV-1 infected individuals were first cultured alone or in the presence of anti-Vβ1CAR-iNKT, anti-Vβ2CAR-iNKT, and anti-CD19 CAR-iNKT cells for 18 hours, and then the frequency of CD4+T cells expressing Tax was assessed by intracellular staining. Although there is clear evidence that CAR mediates the exhaustion of T cells expressing the target TCRVβ molecule, the frequency of Tax+CD4+T cells in any CAR-iNKT co-culture did not change compared with cells cultured alone ( Fig.11 C and 11D, Fig.15 A and 15B).

[0425] Therefore, CAR-iNKT cells targeting the TCRVβ chain do not impair CTL immunity to viral antigens nor promote the replication activity of HTLV-1, both of which are important considerations for the safe treatment of TCL and ATL.

[0426] Embodiment 7: With (G 4 S)2 and (G 4 Comparison of CAR constructs with S)3 linkers

[0427] In order to compare the effect of linker length on in vitro CAR efficacy, 4 S)2 or (G 4 PBMC T cells were transduced with TCRVβ1CAR and TCRVβ2CAR expressing S)3 linker and co-cultured with primary T cell target cells expressing TCRVβ1 or TCRVβ2.

[0428] discuss

[0429] The present invention demonstrates that anti-TCRVβCAR immunotherapy can be developed as a sensitive, specific and efficient strategy for treating T cell malignancies expressing TCRVβ. Given that the distribution of the TCRVβ library in T cell malignancies is the same as that in normal T cells, about 15% of TCLs can be targeted by the CAR developed and tested herein, laying the foundation for the development of CARs for all TCRVβ families, which have available monoclonal antibodies (currently about 70% of the TCRβ chain family) or can develop monoclonal antibodies. Among the three exemplary TCRVβCARs, the best in vitro killing of TCRVβ1 and TCRVβ2 was observed, while the in vitro killing of targets expressing TCRVβ9 was poor. This phenomenon corresponds to the staining intensity of the corresponding monoclonal antibodies on T cells, indicating that the anti-TCRVβ9 monoclonal antibodies and the corresponding CARs have lower affinity, highlighting the necessity of selecting high-affinity monoclonal antibodies.

[0430] The use of two effector platforms (T cells and iNKT cells) showed strong anti-TCL activity of anti-TCRVβCAR, thus providing the prospect of autologous or allogeneic and off-the-shelf cellular immunotherapy, respectively. The subcutaneous TCL model used to test the in vivo efficacy of TCRVβCAR-iNKT cells is similar to human TCL, in which the skin is often the primary or secondary site of disease. Although the potential toxicity of allogeneic CAR-iNKT cells will ultimately be determined in clinical trials, early clinical experience with allogeneic CAR-iNKT cells for B-cell lymphomas has shown no significant toxicity and aGVHD 30,31 The data also suggest that CAR-iNKT cells would provide additional therapeutic advantages compared to CAR-T cells in cases of T-cell malignancies that co-express TCRVβ and CD1d, such as T-lymphoblastic lymphoma, as exemplified by the Jurkat T-cell line. The addition of αGalCer to the therapeutic armamentarium could further enhance the anti-leukemia / lymphoma effects mediated by CAR-iNKT cells.

[0431] Reassuringly, anti-TCRVβCAR-iNKT immunotherapy did not appear to affect adaptive antiviral immunity: the frequencies of influenza-specific or HTLV-1Tax-specific CD8+ T cells did not change when PBMCs from HTLV-1 carriers were cultured with or without anti-TCRVβCAR-iNKTs. The inventors also evaluated the effect of CAR-iNKT activity on proviral expression of bystander HTLV-1-infected CD4+ T cells and observed no difference in HTLV-1Tax expression with or without CAR-iNKT cells. Therefore, at least in short-term in vitro assays, the inventors ruled out the possibility that bystander activation of HTLV-1-infected T cells favors HTLV-1 proviral reactivation.

[0432] Summarize

[0433] In the present disclosure, the inventors have successfully demonstrated that the novel and innovative anti-TCRVβCAR precision medicine approach disclosed herein for the treatment of TCL and ATL can be applied to all TCRVβ chains. Due to the unique advantages described above, it is preferred to use an allogeneic iNKT cell platform as effector cells, which will enable the rapid deployment of pre-prepared allogeneic anti-TCRVβCAR-iNKT cells in HTLV-1 endemic areas and combined with advanced clinical trial designs (e.g., Bayesian optimal interval design). The ready-made anti-TCRVβCAR-iNKT cells can be tested not only in patients with ATL, but also in individuals infected with HTLV-1. As the inventors recently demonstrated, clonal T cell expansion exceeding a certain threshold indicates a high risk of developing ATL, a highly difficult-to-cure cancer. 32，33 .

[0434] References:

[0435] 1. Cancer Research UK. Cancer Statistics for the UK. Available at: https: / / www.cancerresearchuk.org / health-professional / cancer-statistics-for-the-uk. (Accessed on October 18, 2021)

[0436] 2. Watanabe, T. Current status of HTLV-1infection. Int. J. Hematol. 94, pp. 430–4 (2011).

[0437] 3.Cook,LBM&Taylor,GPHTLV-1: the silent impact revealed.LancetInfect.Dis.(2019).doi:10.1016 / S1473-3099(19)30432-3

[0438] 4. Tembhare, P. et al. Flow cytometric immunophenotypic assessment of T-cell clonality by Vβ repertoire analysis: detection of T-cell clonality at diagnosis and monitoring of minimal residual disease following therapy. Am. J. Clin. Pathol. 135, 890–900 (2011).

[0439] 5. Tembhare, P. et al. Flow cytometric immunophenotypic assessment of T-cell clonality by vβ repertoire analysis in fine-needle aspirates and cerebrospinal fluid. Am. J. Clin. Pathol. 137, 220–6 (2012).

[0440] 6. Rowan, A.G. et al. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells. PLOS Pathog. 12, e1006030 (2016).

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[0442] 8. Fleischer, L.C., Spencer, H.T. & Raikar, S.S. Targeting T cell malignancies using CAR-based immunotherapy: challenges and potential solutions. J. Hematol. Oncol. 12, 141 (2019).

[0443] 9. Maciocia, P. M. et al. Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies. Nat. Med. 23, (2017).

[0444] 10. Nikolich-Zugich, J., Slifka, M. K. & Messaoudi, I. The many important facets of T-cell repertoire diversity. Nat. Rev. Immunol. 4, 123–32 (2004).

[0445] 11. Karadimitris, A. et al. Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl. Acad. Sci. U.S.A. 98, 3294–8 (2001).

[0446] 12. Exley, M. A., Wilson, S. B. & Balk, S. P. Isolation and Functional Use of Human NKT Cells. Curr. Protoc. Immunol. 119, 14.11.1-14.11.20 (2017).

[0447] 13. Godfrey, D. I. & Kronenberg, M. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114, 1379–88 (2004).

[0448] 14. Dellabona, P., Padovan, E., Casorati, G., Brockhaus, M. & Lanzavecchia, A. An invariant V alpha 24-J alpha Q / V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8-T cells. J. Exp. Med. 180, 1171–6 (1994).

[0449] 15. Exley, M. et al. CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes. Immunology 100, 37–47 (2000).

[0450] 16. Kawano, T. et al. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science 278, 1626–9 (1997).

[0451] 17. Brossay, L. et al. CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J. Exp. Med. 188, 1511–8 (1998).

[0452] 18. Slauenwhite, D. & Johnston, B. Regulation of NKT Cell Localization in Homeostasis and Infection. Front. Immunol. 6, 255 (2015).

[0453] 19. Johnston, B., Kim, C. H., Soler, D., Emoto, M. & Butcher, E. C. Differential chemokine responses and homing patterns of murine TCR alpha beta NK T cell subsets. J. Immunol. 171, 2960–2969 (2003).

[0454] 20. Kim, C. H., Johnston, B. & Butcher, E. C. Trafficking machinery of NK T cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NK T cell subsets with distinct cytokine-producing capacity. Blood 100, 11–16 (2002).

[0455] 21. Salio, M., Silk, J. D., Yvonne Jones, E. & Cerundolo, V. Biology of CD1- and MR1-Restricted T Cells. Annu. Rev. Immunol. 32, 323–366 (2014).

[0456] 22. Chaudhry, M. S. & Karadimitris, A. Role and regulation of CD1d in normal and pathological B cells. J. Immunol. 193, 4761–4768 (2014).

[0457] 23. Gumperz, J. E., Miyake, S., Yamamura, T. & Brenner, M. B. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195, 625–636 (2002).

[0458] 24. Lee, P. T. et al. Testing the NKT cell hypothesis of human IDDM pathogenesis. J. Clin. Invest. 110, 793–800 (2002).

[0459] 25. Chaidos, A. et al. Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation. Blood 119, 5030–6 (2012).

[0460] 26. Rubio, M.-T. et al. Pre-transplant donor CD4-invariant NKT cell expansion capacity predicts the occurrence of acute graft-versus-host disease. Leukemia 31, 903–912 (2017).

[0461] 27. Schneidawind, D. et al. Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality. Blood 125, 3491–3500 (2015).

[0462] 28. Mavers, M., Maas-Bauer, K. & Negrin, R. S. Invariant Natural Killer T Cells As Suppressors of Graft-versus-Host Disease in Allogeneic Hematopoietic Stem Cell Transplantation. Front. Immunol. 8, 900 (2017).

[0463] 29. Rotolo, A. et al. Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting. Cancer Cell 34, 596 - 610, e11 (2018).

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[0465] 31. Heczey, A. et al. Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis. Nat. Med. 26, 1686–1690 (2020).

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[0467] 33. Wolf, S. N. et al. Quantification of T cell clonality in human T cell leukaemia virus type-1 carriers can detect the development of adult T cell leukaemia early. Blood Cancer J. 11, 66 (2021).

Claims

1. A chimeric antigen receptor (CAR) construct, wherein: The CAR construct is specific to the variable region (Vβ) subunit of the T cell receptor (TCR) β chain of T cells.

2. The CAR construct according to claim 1, wherein: The CAR construct is specific for pathogenic T cells.

3. The CAR construct according to claim 2, wherein: Pathogenic T cells are (i) malignant, pathogenic T cells, or (ii) non-malignant, pathogenic T cells.

4. A CAR construct according to any one of the preceding claims, wherein: The Vβ subunit is selected from the group of Vβ subunits shown in Table 1.

5. A CAR construct according to any one of the preceding claims, wherein: The CAR construct targets a TCR Vβ subunit on a T cell, wherein the TCR Vβ subunit is selected from the following Vβ subunits: TCR-Vβ1, TCR-Vβ2, TCR-Vβ9, and TCR-Vβ11.

6. A CAR construct according to any one of the preceding claims, wherein: The CAR construct is specific for TCR-Vβ1, and optionally, the TCR-Vβ1 subunit comprises an amino acid sequence substantially as shown in SEQ ID No: 1 or a variant or fragment thereof.

7. A CAR construct according to any one of the preceding claims, wherein: The CAR construct is specific for TCR-Vβ2, and optionally, the TCR-Vβ2 subunit comprises an amino acid sequence substantially as shown in SEQ ID No: 2 or a variant or fragment thereof.

8. A CAR construct according to any one of the preceding claims, wherein: The CAR construct is specific for TCR-Vβ9, and optionally, the TCR-Vβ9 subunit comprises an amino acid sequence substantially as shown in SEQ ID No: 3 or a variant or fragment thereof.

9. A CAR construct according to any one of the preceding claims, wherein: The CAR construct is specific for TCR-Vβ11, and optionally, the TCR-Vβ11 subunit comprises an amino acid sequence substantially as shown in SEQ ID No: 4 or a variant or fragment thereof.

10. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises: a) signal peptide; b) an antigen-binding domain or portion specific for the TCR-Vβ subunit, preferably an anti-TCRVβ antibody or a functional fragment thereof specific for the TCR-Vβ subunit, more preferably a single-chain variable fragment (scFv) domain of an anti-TCRVβ antibody; c) a hinge region, preferably comprising a transmembrane domain, most preferably a hinge region comprising a transmembrane domain and a cytoplasmic region; d) a primary stimulatory domain; and / or e) a co-stimulatory domain, preferably two or more co-stimulatory domains.

11. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises a signal peptide comprising human CD8α or a fragment or variant thereof.

12. The CAR construct according to claim 11, wherein The CAR construct comprises a signal peptide comprising an amino acid sequence substantially as shown in SEQ ID No: 5 or a fragment or variant thereof, and / or the signal peptide is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 6 or a fragment or variant thereof.

13. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises an antigen binding domain or portion specific to the TCR-Vβ subunit, and the antigen binding domain or portion comprises an anti-TCRVβ antibody or a functional fragment thereof specific to the TCR-Vβ subunit. Optionally, the antigen binding domain specific to the TCR-Vβ subunit comprises a single-chain variable fragment (scFv) domain of an anti-TCRVβ antibody.

14. The CAR construct according to claim 13, wherein: The CAR construct comprises an scFv, wherein the scFv comprises VL and / or VH from an anti-TCRVβ1 antibody, and the CAR construct: (i) comprising a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 7 or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 8 or a fragment or variant thereof; and / or (ii) comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 10 or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 11 or a fragment or variant thereof.

15. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises an scFv, wherein the scFv comprises VL and / or VH from an anti-TCRVβ2 antibody, wherein the CAR construct: (i) comprising a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 11 or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 12 or a fragment or variant thereof; and / or (ii) comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 13 or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 14 or a fragment or variant thereof.

16. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises an scFv, wherein the scFv comprises VL and / or VH from an anti-TCRVβ9 antibody, wherein the CAR construct: (i) comprising a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 15 or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 16 or a fragment or variant thereof; and / or (ii) comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 17 or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 18 or a fragment or variant thereof.

17. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises an scFv, wherein the scFv comprises VL and / or VH from an anti-TCRVβ11 antibody, wherein the CAR construct: (i) comprising a VL chain having an amino acid sequence substantially as shown in SEQ ID No: 19 or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 20 or a fragment or variant thereof; and / or (ii) comprises a VH chain having an amino acid sequence substantially as shown in SEQ ID No: 21 or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 22 or a fragment or variant thereof.

18. The CAR construct according to any one of claims 14-17, wherein, The VH and VL sequences are separated by at least one linker sequence, which comprises an amino acid sequence substantially as shown in SEQ ID No: 23 or a fragment or variant thereof.

19. A CAR construct according to any one of the preceding claims, wherein: The CAR construct comprises a hinge domain comprising: (i) an extracellular domain or a portion thereof; (ii) a transmembrane (TM) domain or a portion thereof; and (iii) a cytoplasmic domain or a portion thereof.

20. The CAR construct of claim 19, wherein: (i) the hinge domain comprises a CD8α sequence or a portion thereof, more preferably a human CD8α sequence or a portion thereof; (ii) the hinge domain comprises an amino acid sequence substantially as shown in SEQ ID No: 24 or a fragment or variant thereof; (iii) the hinge comprises or consists essentially of the amino acid sequence shown in SEQ ID No: 25 or a fragment or variant thereof; and / or (iv) the hinge domain is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 26 or a fragment or variant thereof.

21. The CAR construct according to claim 20, wherein The hinge domain comprises or is derived from a human CD8α sequence or a portion thereof, wherein the human CD8α sequence or a portion thereof comprises an amino acid sequence substantially as shown in SEQ ID No: 51 or a fragment or variant thereof.

22. The CAR construct of claim 21, wherein: (i) the hinge domain comprises the extracellular domain of human CD8α or a portion thereof, wherein the extracellular domain or a portion thereof is defined by amino acids 138-182 of human CD8α as shown in SEQ ID No: 51 or a portion thereof; (ii) the hinge domain is at least 1, 2 or 3 amino acids longer at the N-terminus compared to amino acids 138-182 of human CD8α as shown in SEQ ID No:51; (iii) the hinge domain is at least 4, 5 or 6 amino acids longer at the N-terminus compared to amino acids 138-182 of human CD8α as shown in SEQ ID No:51; and / or (iv) the hinge domain is at least 7, 8, 9 or 10 amino acids longer at the N-terminus compared to amino acids 138-182 of human CD8α as shown in SEQ ID No:

51.

23. The CAR construct of claim 22, wherein: (i) the hinge domain comprises the extracellular domain of human CD8α or a portion thereof, wherein the extracellular domain or a portion thereof is defined by amino acids 137-182, 136-182 or 135-182 of human CD8α as shown in SEQ ID No: 51; (ii) the hinge domain comprises the extracellular domain of human CD8α or a portion thereof, wherein the extracellular domain or a portion thereof is defined by amino acids 134-182, 133-182 or 132-182 of human CD8α as shown in SEQ ID No: 51; and / or (iii) the hinge domain comprises the extracellular domain of human CD8α or a portion thereof, wherein the extracellular domain or the portion thereof is defined by amino acids 131-182, 130-182, 129-182 or 128-182 of human CD8α as shown in SEQ ID No:

51.

24. The CAR construct according to claim 21, wherein The hinge domain comprises the cytoplasmic domain of human CD8α or a portion thereof, and the cytoplasmic domain or a portion thereof is defined by amino acids 204-206 of human CD8α as shown in SEQ ID No: 51 or a portion thereof.

25. The CAR construct of claim 24, wherein: (i) the cytoplasmic domain is at least 1 amino acid longer at the C-terminus compared to amino acids 204-206 of human CD8α as shown in SEQ ID No:51; (ii) the cytoplasmic domain is at least 2 amino acids longer at the C-terminus compared to amino acids 204-206 of human CD8α as shown in SEQ ID No:51; (iii) the cytoplasmic domain is at least 3 amino acids longer at the C-terminus compared to amino acids 204-206 of human CD8α as shown in SEQ ID No:51; and / or (iv) the cytoplasmic domain is at least 4 amino acids longer at the C-terminus compared to amino acids 204-206 of human CD8α as shown in SEQ ID No:

51.

26. The CAR construct of claim 25, wherein: (i) the cytoplasmic domain is defined by amino acids 204-207 of human CD8α as shown in SEQ ID No:51; (ii) the cytoplasmic domain is defined by amino acids 204-208 of human CD8α as shown in SEQ ID No:51; (iii) the cytoplasmic domain is defined by amino acids 204-209 of human CD8α as shown in SEQ ID No: 51; and / or (iv) the cytoplasmic domain is defined by amino acids 204-210 of human CD8α as shown in SEQ ID No:

51.

27. The CAR construct according to claim 19, wherein The CAR construct comprises a hinge domain, wherein the hinge domain comprises: (i) a human CD8α extracellular domain or a portion thereof, wherein the human CD8α extracellular domain or the portion thereof has an amino acid sequence substantially as shown in SEQ ID No: 24 or a fragment or variant thereof; (ii) a human CD8α transmembrane (TM) domain or a portion thereof, wherein the human CD8α transmembrane domain or a portion thereof has an amino acid sequence substantially as shown in SEQ ID No: 49 or a fragment or variant thereof; and (iii) a human CD8α cytoplasmic domain or a portion thereof, wherein the human CD8α cytoplasmic domain or the portion thereof has an amino acid sequence substantially as shown in SEQ ID No: 50 or a fragment or variant thereof.

28. A CAR construct according to any one of the preceding claims, wherein, The CAR construct comprises an intracellular domain comprising a co-stimulatory domain that primarily stimulates the CD3ζ chain and CD28.

29. The CAR construct according to claim 28, wherein The CAR construct comprises a costimulatory domain of CD28, the costimulatory domain of CD28 having an amino acid sequence substantially as shown in SEQ ID No: 27 or a fragment or variant thereof, and / or the costimulatory domain of CD28 is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 28 or a fragment or variant thereof.

30. The CAR construct according to claim 28 or 29, wherein: The CAR construct comprises 1 or 2 co-stimulatory domains, which are optionally selected from CD28, 4-1BB signaling domains and OX40 signaling domains.

31. The CAR construct according to any one of claims 28-30, wherein, The CAR construct comprises a stimulatory protein CD3ζ having an amino acid sequence substantially as shown in SEQ ID No: 29 or a fragment or variant thereof, and / or the stimulatory protein CD3ζ is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 30 or a fragment or variant thereof.

32. A CAR construct according to any one of the preceding claims, wherein, The CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 31 or 33, or a fragment or variant thereof; and / or the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 32 or 34, or a fragment or variant thereof.

33. A CAR construct according to any one of the preceding claims, wherein, The CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 35 or 37, or a fragment or variant thereof; and / or the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 36 or 38, or a fragment or variant thereof.

34. A CAR construct according to any one of the preceding claims, wherein, The CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 39 or 41, or a fragment or variant thereof; and / or the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 40 or 42, or a fragment or variant thereof.

35. A CAR construct according to any one of the preceding claims, wherein, The CAR construct comprises an amino acid sequence substantially as shown in SEQ ID No: 43 or 45, or a fragment or variant thereof; and / or the CAR construct is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 44 or 46, or a fragment or variant thereof.

36. A nucleic acid encoding the CAR construct of any one of claims 1-35.

37. An expression vector or plasmid encoding the CAR construct of any one of claims 1-35 or comprising the nucleic acid of claim 36.

38. effector cells, which express the CAR construct of any one of claims 1-35 or comprise the nucleic acid of claim 36 or comprise the vector of claim 37.

39. The effector cell according to claim 38, wherein The effector cells are (i) normal or conventional αβ T cells, or (ii) innate lymphocytes, optionally invariant natural killer T (iNKT) cells, γδ T cells or NK cells.

40. The effector cell according to claim 38 or 39, wherein The effector cells are iNKT cells.

41. A method for producing an effector cell expressing an anti-TCRVβCAR, the method comprising transducing the effector cell with the nucleic acid of claim 29 or the vector of claim 37, so that the effector cell expresses the anti-TCRVβCAR.

42. The method according to claim 41, wherein: The effector cells are (i) normal or conventional αβT cells, or (ii) innate lymphocytes, preferably invariant natural killer T (iNKT) cells, γδT cells or NK cells.

43. The method according to claim 41 or 42, wherein: The method comprises an initial step of isolating effector cells from peripheral blood cells (PBCs) and activating the effector cells with one or both of CD3 and CD28 antibodies.

44. The method according to any one of claims 41 to 43, wherein: The effector cells are activated with an interleukin, preferably, the interleukin is IL-15.

45. A pharmaceutical composition comprising a therapeutically effective amount of a CAR construct as described in any one of claims 1-35, a nucleic acid as described in claim 29, a vector as described in claim 37, or an effector cell as described in any one of claims 38-40, and a pharmaceutically acceptable excipient.

46. ​​The pharmaceutical composition according to claim 45, wherein The pharmaceutical composition comprises a plurality of effector cells, preferably T cells or iNKT cells, and the composition comprises at least 100, 1000, 10,000, 100,000, 1,000,000 or at least 10,000,000 effector cells.

47. A CAR construct according to any one of claims 1-35, a nucleic acid according to claim 36, a vector according to claim 37, or an effector cell according to any one of claims 38-40, for use in therapy or diagnosis.

48. A CAR construct according to any one of claims 1-35, a nucleic acid according to claim 36, a vector according to claim 37, or an effector cell according to any one of claims 38-40, for use in (i) immunotherapy; (ii) for treating, preventing or ameliorating cancer; (iii) for treating, preventing or ameliorating autoimmune diseases; or (iv) for treating, preventing or ameliorating any disease characterized by the presence of pathogenic T cells.

49. The CAR construct according to any one of claims 1-35, the nucleic acid according to claim 36, the vector according to claim 37 or the effector cell according to any one of claims 38-40, for use according to claim 47 or 48, wherein, The cancer is a T-cell malignancy, which is a solid tumor or a liquid tumor.

50. The CAR construct according to any one of claims 1-35, the nucleic acid according to claim 36, the vector according to claim 37 or the effector cell according to any one of claims 38-40 for use in any one of claims 47-49, wherein, The T-cell malignancy is selected from the following: hematopoietic lymphocytic neoplasms; precursor T-cell neoplasms; T-lymphoblastic leukemia / lymphoma; mature T-cell neoplasms; peripheral T-cell lymphoma (PRCL); mature T-cell leukemia; primary cutaneous T-cell lymphoproliferation and lymphoma; intestinal T-cell lymphoproliferation and lymphoma; hepatosplenic T-cell lymphoma; anaplastic large cell lymphoma; nodal follicular helper T (TFH) cell lymphoma; peripheral T-cell lymphoma, NOS; EBV-positive NK cell and T-cell lymphoma; EBV-positive T-cell lymphoproliferation; subcutaneous panniculitis-like lymphoma (SPTCL); angioimmunoblastic T-cell lymphoma (AITL); and cutaneous T-cell lymphoma (CTCL).

51. The CAR construct according to any one of claims 1-35, the nucleic acid according to claim 36, the vector according to claim 37 or the effector cell according to any one of claims 38-40, for use in claim 47 or 48, wherein, The autoimmune disease is a disease caused by pathogenic autoreactive T cells, or is selected from systemic lupus erythematosus, rheumatoid arthritis and myasthenia gravis.

52. A method for preparing the pharmaceutical composition of claim 45 or 46, the method comprising combining a therapeutically effective amount of the CAR construct of any one of claims 1-35, the nucleic acid of claim 36, the vector of claim 37, or the effector cell of any one of claims 38-40 with a pharmaceutically acceptable carrier.

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