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

Abstract

The present invention relates to chimeric antigen receptors (CARs) themselves, CAR constructs, and T cell receptor (TCR) beta chain variable region beta (TCRVβ) CARs. The invention relates to the use of such CARs in immunotherapy, for example, in treating, preventing, or ameliorating conditions caused by or involving pathogenic T cells, such as cancers, e.g., T cell malignancies, and autoimmune diseases. The invention particularly relates to conventional T cells and invariant natural killer T (iNKT) cells expressing anti-TCRVβ CARs, and methods for their production. The invention extends to nucleic acids and vectors encoding such anti-TCRVβ CARs, as well as pharmaceutical compositions comprising the constructs and cells, and medical uses of such compositions, anti-TCRVβ CAR constructs, and T cells and iNKT cells expressing anti-TCRVβ CARs.

Description

[Technical Field]

[0001] The present invention relates to chimeric antigen receptors (CARs) per se and CAR constructs, particularly, but not exclusively, to T cell receptor (TCR) beta chain variable region beta (TCRVβ) CARs. The invention relates to the use of such CARs in immunotherapy, for example, in treating, preventing, or ameliorating cancers, such as T cell malignancies, and conditions caused by or involving pathogenic T cells, such as autoimmune diseases. The invention particularly relates to conventional T cells and invariant natural killer T (iNKT) cells expressing anti-TCRVβ CARs, and methods for producing the same. The invention extends to nucleic acids and vectors encoding such anti-TCRVβ CARs, as well as pharmaceutical compositions comprising the constructs and cells, and medical uses of such compositions, anti-TCRVβ CAR constructs, and T cells and iNKT cells expressing anti-TCRVβ CARs. [Background technology]

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

[0003] Expression of the cell surface T cell receptor (TCR) α / β chain heterodimer is restricted to 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, which in humans has 23 families and 114 alleles. Each family comprises 0.5–9% of the normal T cell repertoire, with TCRVβ2 being the most common. Signals from the TCR determine cell fate in normal T cells, and components of the TCR signaling pathway are frequently mutated in TCL, including ATL, indicating the driving role of the TCR in TCL carcinogenesis. Surface TCR expression is also expressed on blood and lymph node lymphoma T cells.4~6 as well as stable in non-malignant pathogenic T cells.

[0004] CAR-T therapeutic approaches in B-cell leukemia and lymphoma that target pan-B-cell markers such as CD19 and CD20 can induce durable clinical remissions in up to 40% of patients with relapsed / refractory disease, albeit with clinically tolerable pan-B-cell depletion and hypoglobulinemia. 7 However, pan-T cell depletion results in significant immunosuppression that is clinically intolerable. Because most TCLs are CD4+, anti-CD4 CAR-T therapy has also been proposed for these lymphomas (see, e.g., clinical trial NCT03829540). Unless rescued by stem cell transplantation, such an approach also results in immunosuppression similar to acquired immune deficiency syndrome. Other CAR targets include CD5 and CD7. However, these are widely expressed on healthy T cells and can cause fratricide reactions unless CAR effector cells are further gene-edited to lack expression of these markers. 8 Recently, the effectiveness of using CAR-T cells targeting the TCR β chain constant region 1, which is expressed in approximately 40–60% of TCR αβ lymphocytes, has been reported. 9 The impact of 40–60% depletion of the T cell repertoire on an individual's immune status remains unclear. As reported in mouse models, loss of half of the TCR sequence can severely impair immunity in humans. 10 Current anti-T cell lymphoma CAR-based immunotherapies eliminate either all or 50% of healthy T cells, thus leaving treated patients with T cell lymphoma severely or significantly immunosuppressed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Watanabe, T. Int. J. Hematol. 94, 430 - 4 (2011)

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Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, there is a need to provide improved immunotherapies for T-cell malignancies such as T-cell lymphomas, including ATL, and for treating diseases such as certain autoimmune diseases caused directly or indirectly by pathogenic non-malignant T cells, as well as for targeting normal non-pathogenic T cells. [Means for solving the problem]

[0007] To combat malignant and non-malignant pathogenic and non-pathogenic T cells, we developed CAR-iNKT (invariant natural killer T) cells as an "off-the-shelf" (readily available) allogeneic immunotherapy. iNKT cells are rare (i.e., they account for less than 0.1% of the T cell population) and are an evolutionarily conserved subset of T cells that share characteristics of the innate and adaptive immune responses. 11~13 In humans, iNKTs are characterized by the expression of the invariant TCR Vα24Jα18 chain, which is mostly paired with the divergent 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 extralymphoid tissues. 18~20 , where it regulates various immune responses, including anti-tumor and anti-pathogen responses. 21、22 .

[0008] iNKT cells protect against acute graft-versus-host disease (aGVHD) 25~28 Therefore, CAR-iNKT cell immunotherapy can be delivered from allogeneic healthy donors as an "off-the-shelf" treatment without the need for deletion of endogenous TCRs as is the case with conventional T cells. Conversely, standard autologous CAR-T immunotherapy can be limited by financial and operational challenges and suboptimal compatibility of patient-derived T cells.

[0009] Based on the above considerations, we explored targeting the ATL / TCL-specific TCR Vβ chain by CAR engineering of T cells and iNKT cells. To address the urgent clinical need for novel therapeutic approaches, we developed an exemplary chimeric antigen receptor (CAR) that targets a group of subunits of the T cell receptor (TCR) β chain constitutively expressed by mature T cell leukemias and lymphomas.

[0010] Thus, in a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR) construct specific for the T cell receptor (TCR) beta chain variable region (Vβ) subunit of a T cell.

[0011] As shown in the Examples, the inventors have developed various embodiments of exemplary CARs specific for several TCR Vβ subunits of the invention (referred to herein as clone BL37.2 for TCR Vβ1, MPB2D5 for TCR Vβ2, FIN9 for TCR Vβ9, and C21 for TCR Vβ11). Advantageously, using conventional T cells and innate natural killer (iNKT) cells as CAR effector cells, the inventors have surprisingly demonstrated that anti-Vβ CAR-T cells and CAR-iNKT cells bearing CARs of the invention can successfully kill in vitro-expanded primary T cells and ex vivo adult T-cell leukemia (ATL) cells expressing the corresponding TCR Vβ subunit, with surprisingly minimal off-target killing of healthy T cells expressing other TCR Vβ subunits. We successfully demonstrated that CAR-iNKT activity was significantly enhanced when challenged with CD1d-expressing targets pulsed with α-galactosylceramide, a selective ligand for iNKT cells, leading to iNKT cell activation. Ligation of an anti-Vβ CAR induced degranulation, IFN-γ and TNF-α secretion, and upregulation of perforin and granzyme expression by T and iNKT effectors. Targeting T cells expressing a single Vβ subunit spared virus-specific CTLs. Furthermore, in a short in vitro assay, we demonstrated that CAR-mediated killing did not affect the expression of human T-cell leukemia virus type 1, the virus that causes ATL. Finally, anti-TCRVβ CAR-iNKT (but not anti-CD19 CAR-iNKT) significantly (p<0.01) reduced tumor size in a subcutaneous model of T-cell lymphoma. Tumor-bearing mice that received iNKT effectors showed no signs of graft-versus-host disease.

[0012] Thus, through these experiments, we surprisingly demonstrated that CARs targeting the TCR Vβ subunit can efficiently kill malignant T cell clones with minimal off-target cytotoxicity. The CAR-iNKT effector exhibited enhanced killing activity in the presence of iNKT ligands and reduced the risk of acute graft-versus-host disease, making it suitable for off-the-shelf use in third-party donor infusion.

[0013] In one embodiment, the CAR construct is specific for the T cell receptor (TCR) beta chain variable region (Vβ) subunit of normal, non-pathogenic T cells.

[0014] However, in another embodiment, the CAR construct is specific for the T cell receptor (TCR) beta chain variable region (Vβ) subunit of the pathogenic T cell.

[0015] Pathogenic T cells are clonal in nature, and tumor cells share expression of a single TCR Vβ subunit, with no bias or preference in the use of the TCR Vβ subunit family. 4~6 Advantageously, clonally targeting specific TCR Vβ subunits expressed by pathogenic T cells provides a highly selective and tumor-specific therapeutic strategy with "on-target off-tumor" toxicity limited to less than 5-9% of normal T cells. Because expression of each TCR Vβ subunit is restricted to less than 9% of healthy T cells, this approach avoids the treatment-induced immune suppression and immune dysregulation that can be caused by alternative CAR-based immunotherapies for ATL / TCL. This is because the novel construct preserves more than 90% of healthy T cells.

[0016] Therefore, based on the above considerations, we explored targeting the ATL / TCL-specific TCR Vβ chain by CAR engineering of T cells and iNKT cells. To address the urgent clinical need for novel therapeutic approaches, we 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.

[0017] Therefore, preferably, the CAR construct of the present invention is specific for the TCR Vβ subunit of pathogenic T cells.

[0018] "Pathogenic T cells" can refer to T cells expressed in clinical disorders such as T cell malignancies, including TCL and ATL, infectious diseases, and autoimmune diseases. Thus, anti-TCRVβ CARs can highly selectively target pathogenic T cells, such as those involved in T cell lymphomas and T cell leukemias, as well as T cells that cause autoimmune diseases.

[0019] Thus, the pathogenic T cells may be malignant pathogenic T cells. Alternatively, the pathogenic T cells may be non-malignant pathogenic T cells.

[0020] Healthy T cells express 23 different families of TCR Vβ chain molecules.

[0021] Advantageously, each T cell expresses only one of the TCR Vβ chains. Thus, pathological T cells, such as those in cancers, i.e., T cell lymphomas and T cell leukemias, and those causing autoimmune diseases, express only one type of TCR Vβ chain. However, the same TCR Vβ chain expressed in pathological T cells is also expressed in less than 10% of normal healthy T cells. Therefore, an anti-TCR Vβ CAR that selectively targets pathological T cells will also target less than 10% of the patient's healthy T cells, while leaving more than 90% of the patient's healthy T cells and T cell-dependent immunity intact. Thus, in some embodiments, healthy T cells may be targeted by the CAR construct of the present invention.

[0022] Table 1 below lists the TCR Vβ subunits on T cells along with the associated encoding genes, any one or more of which may be targeted by the CAR constructs of the present invention.

[0023] [Table 1(1)] [Table 1(2)] [Table 1(3)] [Table 1(4)]

[0024] Preferably, therefore, the Vβ subunit may be selected from the group of Vβ subunits shown in Table 1.

[0025] The frequency of the TCR Vβ chain family in T cell lymphomas is similar to that in the normal T cell repertoire shown in Table 1. In a preferred embodiment, the CAR construct targets a TCR Vβ subunit on the T cell, which is any one of the subunits in the left column of Table 1.

[0026] As described in the Examples, the inventors have generated four embodiments of CAR constructs specific for TCR-Vβ1, TCR-Vβ2, TCR-Vβ9, and TCR-Vβ11, respectively. Thus, preferably, the CAR construct targets a TCR Vβ subunit on T cells selected from the group consisting of the following Vβ subunits: TCR-Vβ1, TCR-Vβ2, TCR-Vβ9, and TCR-Vβ11.

[0027] Thus, preferably, the CAR construct is specific for TCR-Vβ1. (Three alleles, TRBV9 * 01~ *One embodiment of the polypeptide sequence of the Homo sapiens (H. sapiens) TCRBV9 (encoded by UniProtKB: A0A0B4J1U6) TCR Vβ1 subunit in which nucleotide sequence 039 is present is represented herein as SEQ ID NO: 1, as follows: [ka]

[0028] Preferably, therefore, the CAR construct is specific for a TCR-Vβ1 subunit comprising an amino acid sequence substantially as set forth in SEQ ID NO: 1, or a variant or fragment thereof.

[0029] Preferably, the CAR construct is specific for TCR-Vβ2. (7 alleles, TRBV20-1 * 01~ * One embodiment of the polypeptide sequence of the TCR Vβ2 subunit of Homo sapiens TCRBV20-1 (encoded by UniProtKB:A0A075B6N2), in which 07 is present, is represented herein as SEQ ID NO:2, as follows: [ka]

[0030] Preferably, therefore, the CAR construct is specific for a TCR-Vβ2 subunit comprising an amino acid sequence substantially as set forth in SEQ ID NO: 2, or a variant or fragment thereof.

[0031] Preferably, the CAR construct is specific for TCR-Vβ9. (Two alleles, TRBV3-1 * 01 and * One embodiment of the polypeptide sequence of the Homo sapiens TCR Vβ9 subunit (TCRBV3-1 - encoded by UniProtKB / Swiss-Prot:A0A576) in which 02 is present is represented herein as SEQ ID NO: 3, as follows: [ka]

[0032] Preferably, therefore, the CAR construct is specific for a TCR-Vβ9 subunit comprising an amino acid sequence substantially as set forth in SEQ ID NO: 3, or a variant or fragment thereof.

[0033] Preferably, the CAR construct is specific for TCR-Vβ11 (one allele present in Homo sapiens TCRBV25-1 * 01 - One embodiment of the polypeptide sequence of the TCR Vβ11 subunit (encoded by UniProtKB:A0A075B6N4) is represented herein as SEQ ID NO: 4, as follows: [ka]

[0034] Preferably, therefore, the CAR construct is specific for a TCR-Vβ11 subunit comprising an amino acid sequence substantially as set forth in SEQ ID NO: 4, or a variant or fragment thereof.

[0035] Preferably, in one embodiment, the CAR construct comprises: a) signaling peptides, b) an antigen-binding domain or antigen-binding 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) preferably a transmembrane domain, most preferably a hinge region or hinge domain comprising a transmembrane domain and a cytoplasmic region; d) a primary stimulatory (or signaling) domain, and / or e) a costimulatory domain, preferably two or more costimulatory domains Includes.

[0036] Surprisingly, the inventors demonstrated that the combination of each of these components generates anti-TCRVβ CAR constructs with highly selective activity against the cognate TCRVβ family chain and significant activity against T-cell lymphoma and leukemia in vitro and in vivo.

[0037] Thus, preferably, the CAR construct comprises a signaling peptide. Advantageously, the signaling peptide is configured to direct the CAR (i.e., being a fusion protein) to the outer membrane of the effector cell (i.e., T cell) expressing the CAR construct. The signaling peptide of the CAR construct may be a native immunoglobulin gene signaling peptide. However, preferably, the signaling 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.

[0038] In one embodiment, the human CD8a signaling peptide can have the amino acid sequence referred to herein as SEQ ID NO:5, as follows: [ka]

[0039] Preferably, therefore, the CAR construct comprises a signaling peptide having an amino acid sequence substantially as set forth in SEQ ID NO: 5, or a fragment or variant thereof.

[0040] In one embodiment, the nucleotide sequence encoding the signaling peptide is referred to herein as SEQ ID NO:6, as follows: [ka]

[0041] Preferably, therefore, the signalling peptide is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 6, or a fragment or variant thereof.

[0042] Preferably, the antigen-binding domain or antigen-binding portion specific for the TCR-Vβ subunit comprises an anti-TCR-Vβ antibody or a functional fragment thereof specific for the TCR-Vβ subunit. More preferably, the antigen-binding domain specific for the TCR-Vβ subunit comprises a single-chain variable fragment (scFv) domain of an anti-TCR-Vβ antibody. Preferably, the antigen-binding domain is located at the C-terminus of the signaling peptide.

[0043] Those skilled in the art will appreciate that an scFv is a fragment of a given antibody consisting of the variable region (V H ) and the light chain variable region (V L In the context of the present invention, the antibody is an antibody specific for the TCR Vβ subunit.

[0044] As discussed herein, there are 23 different families of TCR Vβ chain molecules. It will be understood that any human TCR Vβ chain may be targeted by the CAR constructs of the present invention. TCR β chain sequences are readily available in public databases. For example, any of the TCR Vβs listed in Tables 1 and 2, which provide an overview of TCR genes (TRBVs) and proteins (TCR Vβs) and indicate proteins for which monoclonal antibodies are available or can be developed, may be targeted by the CAR construct. Thus, in one embodiment, the scFv domain of the anti-TCR Vβ antibody is selected from any one of the TCR Vβ chain genes and proteins listed in Tables 1 and 2.

[0045] As shown in Figures 1 and 2, the CAR construct may comprise an scFv, which may comprise a VL (variable light chain) sequence and a VH (variable heavy chain) sequence. Preferably, the VL sequence is upstream (i.e., 5' or N-terminal) of the VH sequence. However, in some embodiments, the VH sequence may be upstream of the VL sequence. Preferably, the VH and VL coding sequences are separated in either orientation by a linker sequence, such as a G4S linker sequence. Preferably, the linker sequence is flexible.

[0046] TCRVβ1 In one preferred embodiment, the CAR construct may comprise an scFv comprising a VL and / or VH derived from an anti-TCRVβ1 antibody. The CAR construct preferably comprises an scFv comprising a VL and VH derived 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 may comprise a light chain variable region and a heavy chain variable region for binding to the TCRVβ1 antigen.

[0047] In one embodiment, the light chain of the scFv of the anti-TCRVβ1 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 7, as follows: [ka]

[0048] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 7, or a fragment or variant thereof.

[0049] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ1 antibody is referred to herein as SEQ ID NO: 8, as follows: [ka]

[0050] Preferably, therefore, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 8, or a fragment or variant thereof.

[0051] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ1 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 9, as follows: [ka]

[0052] Preferably, therefore, the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0053] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ1 antibody is referred to herein as SEQ ID NO: 10, as follows: [ka]

[0054] Preferably, therefore, the VH chain is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 10, or a fragment or variant thereof.

[0055] TCRVβ2 In another preferred embodiment, the CAR construct may comprise an scFv comprising a VL and / or VH derived from an anti-TCRVβ2 antibody. The CAR construct preferably comprises an scFv comprising a VL and VH derived 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 may comprise a light chain variable region and a heavy chain variable region for binding to the TCRVβ2 antigen.

[0056] In one embodiment, the light chain of the scFv of the anti-TCRVβ2 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 1, as follows: [ka]

[0057] Preferably, therefore, the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 11, or a fragment or variant thereof.

[0058] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ2 antibody is referred to herein as SEQ ID NO: 12, as follows: [ka]

[0059] Preferably, therefore, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 12, or a fragment or variant thereof.

[0060] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ2 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 13, as follows: [ka]

[0061] Preferably, therefore, the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 13, or a fragment or variant thereof.

[0062] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ1 antibody is referred to herein as SEQ ID NO: 14, as follows: [ka]

[0063] Preferably, therefore, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 14, or a fragment or variant thereof.

[0064] TCRVβ9 In yet another preferred embodiment, the CAR construct may comprise an scFv comprising a VL and / or VH derived from an anti-TCRVβ9 antibody. The CAR construct preferably comprises an scFv comprising a VL and VH derived from an anti-TCRVβ9 antibody. The VL and VH sequences may, in one embodiment, be derived from FIN9 (i.e., the hybridoma clone name of the anti-TCRVβ9 monoclonal antibody) and may comprise a light chain variable region and a heavy chain variable region for binding to the TCRVβ9 antigen.

[0065] In one embodiment, the light chain of the scFv of the anti-TCRVβ9 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 15, as follows: [ka]

[0066] Therefore, preferably, the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 15, or a fragment or variant thereof.

[0067] In one embodiment, the nucleotide sequence encoding the VL of the scFv of the anti-TCRVβ9 antibody is referred to herein as SEQ ID NO: 16, as follows: [ka]

[0068] Preferably, therefore, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 16, or a fragment or variant thereof.

[0069] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ9 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 17, as follows: [ka]

[0070] Preferably, therefore, the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 17, or a fragment or variant thereof.

[0071] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ9 antibody is referred to herein as SEQ ID NO: 18, as follows: [ka]

[0072] Preferably, therefore, the VH chain is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 18, or a fragment or variant thereof.

[0073] TCRVβ11 In yet another preferred embodiment, the CAR construct may comprise an scFv comprising a VL and / or VH derived from an anti-TCRVβ11 antibody. The CAR construct preferably comprises an scFv comprising a VL and VH derived 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 may comprise a light chain variable region and a heavy chain variable region for binding to the TCRVβ11 antigen.

[0074] In one embodiment, the light chain of the scFv of the anti-TCRVβ11 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 19, as follows: [ka]

[0075] Preferably, therefore, the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 19, or a fragment or variant thereof.

[0076] In one embodiment, the nucleotide sequence encoding the VL chain of the scFv of the anti-TCRVβ11 antibody is referred to herein as SEQ ID NO: 20, as follows: [ka]

[0077] Preferably, therefore, the VL chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 20, or a fragment or variant thereof.

[0078] In one embodiment, the heavy chain of the scFv of the anti-TCRVβ11 antibody can have the amino acid sequence referred to herein as SEQ ID NO: 21, as follows: [ka]

[0079] Preferably, therefore, the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 21, or a fragment or variant thereof.

[0080] In one embodiment, the nucleotide sequence encoding the VH of the scFv of the anti-TCRVβ11 antibody is referred to herein as SEQ ID NO: 22, as follows: [ka]

[0081] Preferably, therefore, the VH chain is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 22, or a fragment or variant thereof.

[0082] 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, in either orientation, are separated by a linker sequence. In one embodiment, the linker sequence may comprise at least one G4S linker sequence, which may be referred to herein as SEQ ID NO: 23, as follows: [ka]

[0083] Preferably, therefore, the CAR construct comprises a linker sequence comprising substantially the amino acid sequence set forth in SEQ ID NO: 23, or a fragment or variant thereof.

[0084] In other embodiments, the linker sequence may comprise multiple repeats of the G4S linker sequence, for example, the linker sequence may comprise two or three repeats of the G4S linker sequence (i.e., 2xG4S or 3xG4S).

[0085] The CAR construct preferably comprises a hinge domain. Preferably, the hinge domain comprises (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 comprises (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 the VH chain.

[0086] 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 CD8α-derived hinge significantly reduces the toxicity of the CAR construct compared to a shorter CD8α-derived hinge. Thus, preferably, in one embodiment, the hinge domain comprises or consists essentially of the full length or portion of human CD8α.

[0087] In one embodiment, the amino acid sequence of human CD8α is referred to herein as SEQ ID NO: 51, as follows: [ka]

[0088] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including at least a portion of the extracellular domain. In one embodiment, the amino acid sequence of the human CD8α extracellular domain or a portion thereof is referred to herein as SEQ ID NO: 24, as follows: [ka]

[0089] Preferably, therefore, the CAR construct comprises a hinge domain comprising the amino acid sequence substantially as set forth in SEQ ID NO: 24, or a fragment or variant thereof.

[0090] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including the complete transmembrane helical domain. In one embodiment, the amino acid sequence of the human CD8α complete transmembrane domain is referred to herein as SEQ ID NO: 49, as follows: [ka]

[0091] Preferably, therefore, the CAR construct comprises or consists of a hinge domain comprising the amino acid sequence substantially as set forth in SEQ ID NO: 49, or a fragment or variant thereof.

[0092] Preferably, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including 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 referred to herein as SEQ ID NO: 50, as follows: [ka]

[0093] Preferably, therefore, the CAR construct comprises or consists of a hinge domain comprising the amino acid sequence substantially as set forth in SEQ ID NO: 50, or a fragment or variant thereof.

[0094] 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 of human CD8α, i.e., the extracellular domain, represented by SEQ ID NO: 51. Preferably, a hinge comprising a portion of the human CD8α extracellular domain, the human CD8α complete transmembrane domain, and a portion of the human CD8α cytoplasmic domain is defined by amino acids 128-210 of human CD8α, which can have the amino acid sequence referred to herein as SEQ ID NO: 25, as follows: [ka]

[0095] Preferably, therefore, the CAR construct comprises or consists of a hinge domain having an amino acid sequence substantially as set forth in SEQ ID NO: 25, or a fragment or variant thereof.

[0096] Advantageously, SEQ ID NO: 25 comprises 55 amino acids from the extracellular domain (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD - SEQ ID NO: 24), the complete transmembrane helical domain (IYIWAPLAGTCGVLLLSLVIT - SEQ ID NO: 49), and 7 amino acids of the cytoplasmic domain (LYCNHRN - SEQ ID NO: 50). This arrangement advantageously confers on the CAR construct a lower risk of cytokine release syndrome and neurotoxicity, side effects commonly associated with CAR-T therapy, without adversely affecting the efficacy of the construct.

[0097] In one embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including the extracellular domain of human CD8α defined by amino acids 138 to 182 of human CD8α as represented by SEQ ID NO: 51.

[0098] Preferably, a CD8α-derived hinge 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 to 182 of human CD8α represented by SEQ ID NO: 51. Therefore, preferably, the CD8α-derived hinge comprises the extracellular domain of human CD8α defined by amino acids 137 to 182, 136 to 182, or 135 to 182 of human CD8α represented by SEQ ID NO: 51.

[0099] However, more preferably, the CD8α-derived hinge 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 to 182 of human CD8α represented by SEQ ID NO: 51. Therefore, more preferably, the CD8α-derived hinge comprises the extracellular domain of human CD8α defined by amino acids 134 to 182, 133 to 182, or 132 to 182 of human CD8α represented by SEQ ID NO: 51.

[0100] Most preferably, the CD8α-derived hinge 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 represented by SEQ ID NO: 51. Therefore, most preferably, the CD8α-derived hinge comprises the extracellular domain of human CD8α defined by amino acids 131-182, 130-182, 129-182, or 128-182 of human CD8α represented by SEQ ID NO: 51.

[0101] In one embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including the transmembrane domain of human CD8α defined by amino acids 183 to 203 of human CD8α, as represented by SEQ ID NO: 51.

[0102] In another embodiment, the CAR construct comprises or consists of a hinge domain derived from human CD8α, including the cytoplasmic domain of human CD8α defined by amino acids 204-206 of human CD8α.

[0103] Preferably, the CD8α-derived hinge comprising the cytoplasmic domain of human CD8α is at least one amino acid longer at the C-terminus than amino acids 204 to 206 of human CD8α represented by SEQ ID NO: 51. Therefore, preferably, the CD8α-derived hinge comprises the cytoplasmic domain of human CD8α defined by amino acids 204 to 207 of human CD8α represented by SEQ ID NO: 51.

[0104] More preferably, the CD8α-derived hinge comprising the cytoplasmic domain of human CD8α is at least two amino acids longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO: 51. Therefore, more preferably, the CD8α-derived hinge comprises the cytoplasmic domain of human CD8α defined by amino acids 204-208 of human CD8α represented by SEQ ID NO: 51.

[0105] Even more preferably, the CD8α-derived hinge 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 represented by SEQ ID NO:51.

[0106] Therefore, even more preferably, the CD8α-derived hinge comprises the cytoplasmic domain of human CD8α defined by amino acids 204 to 209 of human CD8α represented by SEQ ID NO:51.

[0107] Most preferably, the CD8α-derived hinge comprising the cytoplasmic domain of human CD8α is at least four amino acids longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO: 51. Thus, most preferably, the CD8α-derived hinge comprises the cytoplasmic domain of human CD8α defined by amino acids 204-210 of human CD8α represented by SEQ ID NO: 51.

[0108] In one embodiment, the nucleotide sequence encoding the hinge domain is referred to herein as SEQ ID NO:26, as follows: [ka]

[0109] Preferably, therefore, the construct comprises a hinge domain substantially encoded by the nucleotide sequence set forth in SEQ ID NO: 26, or a fragment or variant thereof.

[0110] The CAR construct preferably comprises a primary stimulatory (or signaling) CD3 ζ chain and / or a costimulatory (or signaling) domain of CD28, more preferably an intracellular domain comprising the costimulatory / signaling domain of CD28 and a stimulatory CD3 ζ chain. It will be understood that these components form the basis of second-generation CARs and are required to trigger intracellular signaling pathways. Preferably, the intracellular domain is located 3' to the sequence encoding the hinge domain. The CD28 costimulatory domain may be at the N-terminus of the CD3 ζ chain. Advantageously, the CD28 costimulatory domain significantly improves the efficacy of CD19 CAR T cells in patients with B-cell lymphoma and B-cell acute leukemia, providing TCRV-CARs with significant efficacy against T-cell lymphoma and leukemia in vitro and in vivo.

[0111] In another embodiment, the CAR construct may comprise one or two costimulatory domains, which may be selected from CD28, 4-1BB signaling domain, and OX40 signaling domain.

[0112] One embodiment of a 4-1BB signaling domain can have the amino acid sequence designated herein as SEQ ID NO:46, as follows: [ka]

[0113] Preferably, therefore, the construct comprises a nucleotide sequence encoding the amino acid sequence substantially as set forth in SEQ ID NO: 46, or a fragment or variant thereof.

[0114] In one embodiment, the 4-1BB signaling domain may be encoded by the nucleic acid sequence designated herein as SEQ ID NO:47, as follows: [ka]

[0115] Preferably, therefore, the construct comprises a nucleotide sequence substantially as set out in SEQ ID NO: 47, or a fragment or variant thereof.

[0116] Therefore, in one embodiment, the costimulatory domain of CD28 is referred to herein as SEQ ID NO:27, as follows: [ka]

[0117] Preferably, therefore, the CAR construct comprises the costimulatory domain of CD28 having an amino acid sequence substantially as set forth in SEQ ID NO: 27, or a fragment or variant thereof.

[0118] In one embodiment, the nucleotide sequence encoding the costimulatory domain of CD28 is referred to herein as SEQ ID NO:28, as follows: [ka]

[0119] Preferably, therefore, the costimulatory domain of CD28 is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 28, or a fragment or variant thereof.

[0120] In a preferred embodiment, the CAR construct further comprises the stimulatory protein CD3ζ. In one embodiment, the stimulatory protein CD3ζ can have the amino acid sequence referred to herein as SEQ ID NO: 29, as follows: [ka]

[0121] Preferably, therefore, the CAR construct comprises the stimulatory protein CD3ζ having an amino acid sequence substantially as set forth in SEQ ID NO: 29, or a fragment or variant thereof.

[0122] In one embodiment, the nucleotide sequence encoding the stimulatory protein CD3ζ is referred to herein as SEQ ID NO: 30, as follows: [ka]

[0123] Preferably, therefore, the stimulatory protein CD3ζ is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 30, or a fragment or variant thereof.

[0124] Thus, in a preferred embodiment, the intracellular domain of the CAR construct of the first aspect comprises both the costimulatory domain and the CD3ζ stimulatory domain of CD28.

[0125] It is understood that the position of each component in the CAR constructs described herein is interchangeable. Figure 1 provides a schematic map of an exemplary embodiment of an anti-TCRVβ CAR construct.

[0126] Thus, in a preferred embodiment, the CAR construct comprises each of the elements in the following order: 5' / N-terminal variable light chain of anti-TCRVβ antibody-variable heavy chain of anti-TCRVβ antibody-hinge-co-stimulatory domain comprising CD28-3' / C-terminal stimulatory protein CD3ζ. The use of N-terminal and C-terminal (and 5' and 3') indicates that a feature is either upstream or downstream in the construct (protein or DNA, respectively), and is not intended to indicate that a feature is necessarily a terminal feature.

[0127] More preferably, the CAR construct comprises a 5' / N-terminal signal peptide-variable light chain of an anti-TCRVβ antibody-linker-variable heavy chain of an anti-TCRVβ antibody-hinge-costimulatory domain comprising CD28-3' / C-terminal stimulatory protein CD3ζ.

[0128] "BL37.2 CAR" In one preferred embodiment, the anti-TCRVβ CAR construct is an anti-TCRVβ1 CAR (known herein as "BL37.2 CAR"). Preferably, the anti-TCRVβ1 CAR comprises a 2xG4S linker. In one embodiment, the anti-TCRVβ1 CAR (with a 2xG4S linker) has the amino acid sequence referred to herein as SEQ ID NO: 31, as follows: [ka]

[0129] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 31, or a fragment or variant thereof.

[0130] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ1 CAR (with 2×G4S linkers) is referred to herein as SEQ ID NO: 32, as follows: [ka]

[0131] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 32, or a fragment or variant thereof.

[0132] In another preferred embodiment, the anti-TCRVβ1 CAR construct (known as the "BL37.2 CAR") comprises a 3xG4S linker. In one embodiment, the anti-TCRVβ1 CAR (with a 3xG4S linker) can have the amino acid sequence referred to herein as SEQ ID NO: 33, as follows: [ka]

[0133] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 33, or a fragment or variant thereof.

[0134] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ1 CAR (with a 3×G4S linker) is referred to herein as SEQ ID NO: 34, as follows: [ka]

[0135] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 34, or a fragment or variant thereof.

[0136] "MPB2D5 CAR" In yet another preferred embodiment, the anti-TCRVβ CAR construct is an anti-TCRVβ2 CAR (known as "MPB2D5 CAR"). Preferably, the anti-TCRVβ2 CAR comprises a 2xG4S linker. In one embodiment, the anti-TCRVβ2 CAR (with a 2xG4S linker) has the amino acid sequence designated herein as SEQ ID NO: 35, as follows: [ka]

[0137] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 35, or a fragment or variant thereof.

[0138] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ2 CAR (with 2×G4S linkers) is referred to herein as SEQ ID NO: 36, as follows: [ka]

[0139] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 36, or a fragment or variant thereof.

[0140] In another preferred embodiment, the anti-TCRV beta 2 CAR construct (known as the "MPB2D5 CAR") comprises a 3xG4S linker. In one embodiment, the anti-TCRV beta 2 CAR (with a 3xG4S linker) can have the amino acid sequence referred to herein as SEQ ID NO: 37, as follows: [ka]

[0141] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 37, or a fragment or variant thereof.

[0142] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ2 CAR (with a 3×G4S linker) is referred to herein as SEQ ID NO: 38, as follows: [ka]

[0143] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 38, or a fragment or variant thereof.

[0144] "FIN9 CAR" In one preferred embodiment, the anti-TCRVβ CAR construct is an anti-TCRVβ9 CAR (known as a "FIN9 CAR"). Preferably, the anti-TCRVβ9 CAR comprises a 2xG4S linker. In one embodiment, the anti-TCRVβ9 CAR construct has the amino acid sequence referred to herein as SEQ ID NO: 39, as follows: [ka]

[0145] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 39, or a fragment or variant thereof.

[0146] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ9 CAR (with 2×G4S linkers) is referred to herein as SEQ ID NO: 40, as follows: [ka]

[0147] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 40, or a fragment or variant thereof.

[0148] In another preferred embodiment, the anti-TCRV beta 9 CAR construct (known as a "FIN9 CAR") comprises a 3xG4S linker. In one embodiment, the anti-TCRV beta 9 CAR (with a 3xG4S linker) can have the amino acid sequence referred to herein as SEQ ID NO: 41, as follows: [ka]

[0149] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 41, or a fragment or variant thereof.

[0150] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ9 CAR (with 3×G4S) is referred to herein as SEQ ID NO: 42, as follows: [ka]

[0151] Preferably, therefore, the construct is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 42, or a fragment or variant thereof.

[0152] "C21 CAR" In yet another preferred embodiment, the anti-TCRVβ CAR construct is an anti-TCRVβ11 CAR (known as a "C21 CAR"). Preferably, the anti-TCRVβ11 CAR comprises a 2xG4S linker. In one embodiment, the anti-TCRVβ11 CAR construct (with a 2xG4S linker) may have the amino acid sequence referred to herein as SEQ ID NO: 43, as follows: [ka]

[0153] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 43, or a fragment or variant thereof.

[0154] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ11 CAR (with 2×G4S linkers) is referred to herein as SEQ ID NO: 52, as follows: [ka]

[0155] Preferably, therefore, the construct is encoded by a nucleotide sequence substantially as set out in SEQ ID NO: 52, or a fragment or variant thereof.

[0156] In another preferred embodiment, the anti-TCRVβ11 CAR construct (known as "C21 CAR") comprises a 3xG4S linker. In one embodiment, the anti-TCRVβ11 CAR construct (with a 3xG4S linker) can have the amino acid sequence referred to herein as SEQ ID NO: 44, as follows: [ka]

[0157] Preferably, therefore, the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 44, or a fragment or variant thereof.

[0158] In one embodiment, the nucleotide sequence encoding the anti-TCRVβ11 CAR (with 3×G4S linker) is referred to herein as SEQ ID NO: 45, as follows: [ka]

[0159] Preferably, therefore, the CAR construct is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 45, or a fragment or variant thereof.

[0160] In a second aspect, there is provided a nucleic acid encoding the CAR construct of the first aspect.

[0161] Preferably, the nucleic acid comprises a promoter operably linked to the sequence encoding the CAR construct. The promoter is preferably located 5' to the sequence encoding the signaling peptide. The promoter drives expression of the CAR construct in a host cell.

[0162] The promoter may be any suitable promoter, including a constitutive promoter, an activatable promoter, an inducible promoter, or a tissue-specific promoter. A constitutive promoter allows a heterologous gene (also called a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factors-1 alpha (hEF1a), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter coupled with CMV early enhancer (CAGG). Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as a physical condition, the microenvironment of the engineered immune effector cell, or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducer), or a combination thereof. In some embodiments, the inducing condition does not induce expression of the endogenous gene in the engineered mammalian cell and / or in the subject receiving the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and an activation state of the engineered mammalian cell.

[0163] In one embodiment, the promoter may be the PGK promoter (EMBL number: A19297.1). In one embodiment, the PGK promoter is referred to herein as SEQ ID NO: 48, as follows: [ka]

[0164] Preferably, therefore, the promoter may comprise a nucleotide sequence substantially as set out in SEQ ID NO: 48, or a fragment or variant thereof.

[0165] In a preferred embodiment, the nucleic acid of the second aspect is selected from any of the nucleic acid sequences described herein.

[0166] In a third aspect, there is provided an expression vector or plasmid encoding the CAR construct of the first aspect or comprising a nucleic acid of the second aspect.

[0167] Preferably, the vector is recombinant. Preferably, the vector is a viral vector, more preferably a retroviral vector, and even more preferably a lentiviral vector. Maps showing the plasmid structures of the vectors of exemplary anti-TCRVβ CAR constructs tested in the present invention are shown in Figures 4-7.

[0168] Preferably, the vector comprises a left (i.e., N-terminal) and / or right (i.e., C-terminal) long terminal repeat sequence (LTR). Preferably, each LTR is located at the N-terminus and / or C-terminus of the construct.

[0169] In a preferred embodiment, the vector comprises 5' / N-terminal LTR-promoter-variable light chain of the scFv domain of an anti-TCRVβ antibody-linker-variable heavy chain of the scFv domain of an anti-TCRVβ antibody-hinge-costimulatory domain comprising CD28-stimulatory protein CD3ζ-3' / C-terminal LTR.

[0170] Preferably, the hinge comprises a CD8α hinge and optionally a transmembrane domain and / or a cytoplasmic domain.

[0171] In a fourth aspect, there is provided an effector cell expressing a CAR construct of the first aspect or comprising a nucleic acid of the second aspect or a vector according to the third aspect.

[0172] The vector may be a lentiviral vector. The vector may be a retroviral vector.

[0173] Preferably, effector cells are innate lymphocytes such as normal or conventional αβ T cells, or invariant natural killer T (iNKT) cells, γδ T cells or NK cells.

[0174] Those skilled in the art are aware that conventional αβ T cells are one of two major types of lymphocytes (B cells are the second type) that 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, through the production of cytokines and effector molecules. In humans, T cells simultaneously control multiple insults throughout the body, maintaining immune homeostasis for decades.

[0175] Thus, in one embodiment, the effector cells of the invention are conventional T cells.

[0176] "Conventional T cells" may be defined as T lymphocytes that express the αβ T cell receptor (TCR) and the co-receptors CD4 or CD8, and that are present in peripheral blood, lymph nodes, and tissues.

[0177] However, in another preferred embodiment, the effector cells are iNKT cells.

[0178] iNKT cells are a subset of immunoregulatory effector T cells that account for less than 0.1% of the total T cell population in humans.

[0179] There are several structural and functional differences between iNKT cells and conventional T cells. In particular, iNKT cells almost always express the invariant Va24Ja18 chain, which pairs with the same distinct TCRβ11 chain. Furthermore, 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. In contrast to conventional T cells, which are restricted by highly polymorphic MHC-presented peptides, iNKT cells require expression of CD1d on thymocytes for selection and activation. Conversely, conventional T cells require expression of MHC molecules on thymic epithelial cells for their selection and activation.

[0180] iNKT cells provide effective immune responses against infectious agents (pathogens), tumors, alloreactivity and autoreactivity, and atherosclerosis. Several preclinical studies have demonstrated the ability of adoptively transferred donor iNKT cells to prevent or even eliminate established experimental acute graft-versus-host disease (aGVHD), a phenomenon of alloreactivity that occurs in the setting of allogeneic hematopoietic stem cell transplantation. aGVHD is primarily driven by donor alloreactive T cells activated in response to disparities in major or minor histocompatibility antigens between the donor and recipient. Consistent with preclinical evidence, several clinical observational studies have demonstrated that higher doses or frequencies of donor iNKT cells transplanted into recipients with peripheral blood stem cell grafts confer significant protection from aGVHD without compromising the graft-versus-tumor effect.

[0181] In humans, iNKT cells are quantitatively and qualitatively altered in various types of tumors, including hematological cancers such as multiple myeloma, and tumor tissue infiltration by iNKT cells appears to confer a favorable prognosis in colorectal cancer.

[0182] Many of the antitumor effects of iNKT cells depend on their ability to be cytolytic, either directly via perforin / granzyme and other cell death pathways against CD1d-expressing tumors, or indirectly via the secretion of large amounts of (interferon-gamma) IFNγ and / or secondary activation of NK cells or conventional T cell-dependent antitumor responses.

[0183] It is understood that the effector cells of the fourth aspect are generated by transducing T cells or iNKT cells with a nucleic acid or vector encoding the CAR construct.

[0184] Accordingly, in a fifth aspect there is provided a method of producing an effector cell that expresses an anti-TCRVβ CAR, the method comprising transducing an effector cell with a nucleic acid according to the second aspect or a vector according to the third aspect such that the effector cell expresses the anti-TCRVβ CAR.

[0185] Preferably, effector cells are innate lymphocytes such as normal or conventional αβ T cells, or invariant natural killer T (iNKT) cells, γδ T cells or NK cells.

[0186] Preferably, the method comprises the initial step of isolating effector cells from peripheral blood cells (PBCs). Preferably, the effector cells are activated with one or both of a CD3 antibody and a CD28 antibody. 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 antibody, CD28 antibody and IL-15.

[0187] In preferred embodiments, the nucleic acid and / or vector encoding the anti-TCRVβ CAR may comprise any of the nucleic acids or vectors described herein.

[0188] In a sixth aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a CAR construct according to the first aspect, a nucleic acid according to the second aspect, a vector according to the third aspect, or an effector cell according to the fourth aspect, and a pharmaceutically acceptable excipient.

[0189] Preferably, effector cells are normal (ie, conventional αβ T cells) or innate lymphoid cells such as invariant natural killer T (iNKT) cells, γδ T cells or NK cells.

[0190] 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, or at least 1,000,000, or at least 10,000,000 effector cells.

[0191] In a seventh aspect, there is provided a CAR construct according to the first aspect, a nucleic acid according to the second aspect, a vector according to the third aspect, an effector cell according to the fourth aspect, or a pharmaceutical composition according to the sixth aspect for use in therapy or diagnosis.

[0192] In an eighth aspect, there is provided a CAR construct according to the first aspect, a nucleic acid according to the second aspect, a vector according to the third aspect, an effector cell according to the fourth aspect, or a pharmaceutical composition according to the sixth aspect for use (i) in immunotherapy, (ii) to treat, prevent or ameliorate cancer, (iii) to treat, prevent or ameliorate an autoimmune disease, or (iv) to treat, prevent or ameliorate any disease characterized by the presence of pathogenic T cells.

[0193] In a ninth aspect, the present invention provides a method of (i) treating, preventing or ameliorating a disease in a subject with immunotherapy, (ii) treating, preventing or ameliorating cancer, (iii) treating, preventing or ameliorating an autoimmune disease in a subject, or (iv) treating, preventing or ameliorating any disease characterized by the presence of pathogenic T cells, the method comprising administering or having administered to a patient in need of such treatment a therapeutically effective amount of a CAR construct according to the first aspect, a nucleic acid of the second aspect, a vector of the third aspect, an effector cell according to the fourth aspect, or a pharmaceutical composition of the sixth aspect.

[0194] In one embodiment of the eighth or ninth aspect of the invention, the CAR construct, effector cell or pharmaceutical composition is for use in treating, preventing or ameliorating cancer. Preferably, the cancer is a T-cell malignancy, which may be a solid or liquid tumor.

[0195] According to the WHO 2022 classification of hematolymphoid neoplasms, T-cell neoplasms may be precursor T-cell neoplasms, such as T-lymphoblastic leukemia / lymphoma or mature T-cell neoplasms, or peripheral T-cell lymphoma (PTCL), all of which have a 5-year survival rate of 35-40% with current treatments. According to the WHO 2022 classification, mature T-cell neoplasms comprise a diverse group of rare, highly malignant diseases in which a patient's T cells become cancerous. These are divided into three categories: nodal, extranodal, and leukemic, each of which is encompassed by the present invention.

[0196] The mature T-cell neoplasm may be a TCL subtype selected from the group consisting of mature T-cell leukemia; primary cutaneous T-cell lymphoid proliferation and lymphoma; intestinal T-cell lymphoid proliferation 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 and lymphoma of childhood, and all specific TCL entities within each of these subtypes according to the WHO classification.

[0197] Adult T-cell leukemia / lymphoma (ATL), more common in Japan and the Caribbean than in the United States, is a rare, aggressive disease associated with human T-cell leukemia virus type 1 (HTLV-1), begins in the liver or spleen, and usually 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.

[0198] Subcutaneous panniculitis-like lymphoma (SPTCL) is the rarest and most ill-defined of the T-cell lymphomas. This lymphoma primarily arises in subcutaneous adipose tissue, where it causes the formation of nodules. Symptoms include fever, chills, weight loss, and oral mucosal ulcers. SPTCL may be either rapidly aggressive or indolent (slow-growing). Treatment includes combination anthracycline-based chemotherapy or local radiation. Precursor T-cell acute lymphoblastic lymphoma or leukemia may be diagnosed as leukemia, lymphoma, or both. This cancer is seen in both children and adults and is most commonly diagnosed in adolescent and adult males. Treatment for newly diagnosed patients with precursor T-cell acute lymphoblastic lymphoma or leukemia is aggressive chemotherapy and radiation therapy. Nelarabine (Arranon®) is approved for the treatment of relapsed or refractory precursor T-cell acute lymphoblastic lymphoma or leukemia in adults and children.

[0199] Angioimmunoblastic T-cell lymphoma (AITL) exemplifies a neoplasm characterized by strong inflammatory and immune responses, as evidenced by its clinical, pathological, cellular, and biological characteristics. Because the tumor cells phenotypically resemble follicular helper T (Tfh) cells, they are thought to function somewhat similarly to non-neoplastic Tfh cells found in reactive (lymphoid) follicular hyperplasia. However, follicles are not hyperplastic but rather depleted or destroyed in the majority of AITL cases. AITL was recently reported to account for 36.1% of PTCL cases.

[0200] Cutaneous T-cell lymphoma (CTCL) accounts for approximately 70-75% of primary cutaneous lymphomas. CTCL may be a CTCL subtype selected from the group consisting of mycosis fungoides (MF), Sézary syndrome (SS), and CD4+ small medium pleomorphic T-cell lymphoproliferative disorder.

[0201] Mycosis fungoides (MF) is the most common subtype. Sézary syndrome (SS) is a more aggressive type of CTCL. Patients with SS have erythroderma (i.e., a rash affecting more than 80% of the body surface area [BSA]), lymphadenopathy, and large numbers of circulating neoplastic CD4+ T cells in the peripheral blood.

[0202] In other embodiments, the CAR construct, effector cell or pharmaceutical composition may be used in treating any disease caused by pathogenic T cells.

[0203] In other embodiments, the CAR construct, effector cell, or pharmaceutical composition may be used in treating, preventing, or ameliorating an autoimmune disease, which may be caused by pathogenic autoreactive T cells or may be selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, and myasthenia gravis.

[0204] It will be understood that the CAR constructs, nucleic acids, vectors, effector cells, or pharmaceutical compositions (collectively referred to herein as "medications") of the present invention may be used for therapy, preferably in monotherapy (e.g., use of the CAR constructs, nucleic acids, vectors, effector cells, or pharmaceutical compositions alone) for use in (i) immunotherapy, (ii) to treat, prevent, or ameliorate cancer, (iii) to treat, prevent, or ameliorate autoimmune disease, or (iv) to treat, prevent, or ameliorate any disease characterized by the presence of pathogenic T cells. Alternatively, the CAR constructs, effector cells, or pharmaceutical compositions of the present invention may be used as an adjunct to or in combination with known immunotherapies, or to treat diseases caused by pathogenic T cells, as well as cancer or autoimmune diseases.

[0205] The agents of the present invention may be combined in compositions having several different forms, depending in particular on the method in which the composition is to be used. Thus, for example, the composition may be in the form of a liquid that is preferably delivered intravenously to a person in need of treatment. It will be understood that the pharmaceutical vehicle of the present invention should be one that is well tolerated by the subject to which it is administered.

[0206] In a preferred embodiment, the agents and medicaments of the present invention may be administered to a subject by injection into the bloodstream or directly to the site in need of treatment. Injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or directly into a tumor.

[0207] It is understood that the amount of CAR construct or vector or effector cell (i.e., drug) required is determined by its biological activity and bioavailability, and therefore depends on the mode of administration, the physiochemical properties of the drug, and whether the drug is used as a monotherapy or in a combination therapy. The frequency of administration is also affected by the in vivo persistence of the drug in the treated subject. The optimal dosage to be administered can be determined by one skilled in the art and will vary depending on the specific drug used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease being treated, such as cancer, T-cell malignancy, or autoimmune disease. Additional factors depending on the specific subject being treated, including the subject's age, weight, sex, diet, and time of administration, will necessitate adjusting the dosage.

[0208] In a tenth aspect, the present invention also provides a process for producing a pharmaceutical composition according to the sixth aspect, the process comprising combining a therapeutically effective amount of a CAR construct according to the first aspect, a nucleic acid according to the second aspect, a vector according to the third aspect, or an effector cell according to the fourth aspect with a pharmaceutically acceptable vehicle.

[0209] A "subject" may be a vertebrate, a mammal, or a domestic animal. Most preferably, the subject is a human.

[0210] A "therapeutically effective amount" of a CAR construct, nucleic acid, vector, or effector cell of a composition is any amount that, when administered to a subject, is the amount of drug necessary to treat the disease being treated, e.g., cancer, or to produce a desired effect.

[0211] 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, or at least 1,000,000, or at least 10,000,000 effector cells are used.

[0212] The "pharmaceutically acceptable vehicle" referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions. Preferably, for successful CAR-effective therapy, the composition containing CAR effector cells is prepared and then delivered as a cell suspension, most preferably intravenously.

[0213] The pharmaceutical vehicle may be liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active agent of the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0214] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The agents may also be prepared as sterile solid compositions that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0215] It will be understood that the present invention also extends to any nucleic acid or peptide, or variant, derivative or analogue thereof, that substantially comprises the amino acid or nucleic acid sequence of any of the sequences referred to herein (including variants or fragments thereof). The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" can refer to a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, such as a sequence that has 40% identity to a sequence specified herein.

[0216] Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65% sequence identity to any of the referenced sequences, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity to any of the referenced sequences, more preferably at least 90% identity to any of the sequences referenced herein, 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.

[0217] Those skilled in the art will understand how to calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences. To calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The identity percentage of two sequences can vary depending on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented by different programs), or structural alignment from 3D comparison, and (ii) the parameters used by the alignment method, such as local versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as function form and constants.

[0218] After alignment, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identical portions may 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. Furthermore, it will be understood that the percentage identity is also strongly dependent on length. Therefore, the shorter the sequence pair, the higher the expected sequence identity will be by chance.

[0219] It will therefore be appreciated 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) is a preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity; for protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet; for DNA and protein alignments: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0220] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T) x 100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps, and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes (i) preparing a sequence alignment using the ClustalW program, for example, using an appropriate set of parameters as described above, and (ii) substituting the values ​​of N and T into the following formula: sequence identity = (N / T) x 100.

[0221] Alternative methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequences set forth herein by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids.

[0222] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein encoded thereby to provide functional variants thereof. Suitable nucleotide variants are those having a sequence that is altered by the substitution of different codons that encode the same amino acid within the sequence, thus resulting in a silent (synonymous) change. Other suitable variants are those that contain all or portions of a sequence that have a homologous nucleotide sequence but are altered by the substitution of different codons that encode amino acids with side chains with similar biophysical properties to the amino acid being replaced, resulting in a conservative change. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is therefore understood that which amino acids may be replaced with amino acids having similar biophysical properties, and one of skill in the art would know the nucleotide sequences encoding these amino acids.

[0223] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. [Brief explanation of the drawings]

[0224] For a better understanding of the present invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which:

[0225] [Figure 1] 1 shows a schematic map of one embodiment of the coding sequence of an anti-TCRVβ CAR lentiviral CAR expression vector of the present invention, which comprises a human CD8α signal peptide, an antigen-binding domain comprising a VL, 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. [Figure 2a] Figure 2a shows the membrane topology of the mature CAR of the present invention. The CD8α signal peptide, VL, linker, and VH domains form the extracellular ectodomain, while the CD8α hinge domain comprises the ectodomain portion, the plasma membrane (PM), and the cytoplasmic fragment. The CD28 costimulatory domain and CD3ζ stimulatory domain form the intracellular endodomain (intracellular region). [Figure 2b] FIG. 2b depicts one embodiment of a CAR of the invention, designated MPB2D5 CAR GS3 CD8283Z (TCRVB 2 CAR). [Figure 3]Figure 3 provides structural details of the design of the coding sequences for each of various embodiments of exemplary anti-TCRVβ CARs of the invention. From top to bottom, the diagram shows a CAR specific for a TCRVβ1 CAR (i.e., anti-TCRVβ1 CAR), a CAR specific for a TCRVβ2 CAR (i.e., anti-TCRVβ2 CAR), a CAR specific for a TCRVβ9 CAR (i.e., anti-TCRVβ9 CAR), and a CAR specific for a TCRVβ11 CAR (i.e., anti-TCRVβ11 CAR). For each embodiment, a construct with a 2x G4S linker and a construct with a 3x G4S linker are shown, respectively. The position of each component of the construct (5' to 3') follows the order disclosed in the schematic map of Figure 1. [Figure 4] Figure 4 shows plasmid maps of the anti-TCRVβ1 CAR (referred to as BL37.2 CAR) construct shown in Figure 3. The top map relates to a first embodiment of the anti-TCRVβ1 CAR that includes a 2xG4S linker, and the bottom map relates to a second embodiment of the anti-TCRVβ1 CAR that includes a 3xG4S linker. [Figure 5] Figure 5 shows plasmid maps of the anti-TCRVβ2 CAR (referred to as MPB2D5 CAR) shown in Figure 3. The top map relates to a first embodiment of the anti-TCRVβ2 CAR that includes a 2xG4S linker, and the bottom map relates to a second embodiment of the anti-TCRVβ2 CAR that includes a 3xG4S linker. [Figure 6] Figure 6 shows the plasmid maps of the anti-TCRVβ9 CAR (referred to as FIN9 CAR) shown in Figure 3. The top map relates to a first embodiment of the anti-TCRVβ9 CAR that includes a 2xG4S linker, and the bottom map relates to a second embodiment of the anti-TCRVβ9 CAR that includes a 3xG4S linker. [Figure 7] Figure 7 shows the plasmid maps of the anti-TCRVβ11 CAR (referred to as C21 CAR) shown in Figure 3. The top map relates to a first embodiment of the anti-TCRVβ11 CAR that includes a 2xG4S linker, and the bottom map relates to a second embodiment of the anti-TCRVβ11 CAR that includes a 3xG4S linker. [Figure 8]Figure 8 shows the specificity, cytokine production, and cytotoxic activity of anti-TCR Vβ CAR-T cells of the invention. Peripheral blood mononuclear cells (PBMCs) from normal donors were cultured alone or transduced with lentiviruses encoding anti-Vβ1, Vβ2, and Vβ9 CAR constructs. Five days after transduction, transduction efficiency was assessed by protein L staining (see Figures 8A and B), and the frequency of CD3+ cells expressing each of the 24 TCR Vβ subunits was quantified (see Figure 8C). Values ​​shown are the average percentage of cells expressing each subunit, normalized to the frequency of cells expressing that subunit in untransduced PBMC controls from two individuals (n=2). Killing of in vitro-expanded autologous primary T cell lines by anti-TCR CAR-T cells and anti-CD19 CAR-T cells (see Figure 8D) and killing of untransduced (UT) and TCR-GFP-transduced (Vβ+) JRT3-T3.5 cells by anti-Vβ CAR-T cells (see Figure 8E). Target cells were stained with CFSE and cocultured in duplicate with effectors at a range of effector-to-target (E:T) ratios. After 24 hours, cells were harvested and stained with 7aad. The frequency of dead targets in each culture condition was assayed by flow cytometry. Results are representative of two or more independent experiments. CD107a recruitment, IFN-γ and TNF-α production by CD3+ anti-Vβ2 and anti-CD19 CAR-T when 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) (see Figure 8F). Results are from a single experiment and are representative of two independent experiments. [Figure 9]Figure 9 shows cytokine production and cytotoxic activity of anti-TCR Vβ CAR-iNKT cells of the present invention. Killing of an in vitro-expanded autologous primary T cell line by anti-TCR CAR-iNKT cells and anti-CD19 CAR-iNKT (see Figure 9A). Killing of untransduced (UT) and TCR-GFP-transduced (TCR-GFP) JRT-3 T3.5 cells by anti-Vβ CAR-iNKT cells (see Figure 9B). Target cells were stained with CFSE and cocultured in duplicate with effectors at a range of effector-to-target (E:T) ratios. After 24 hours, cells were harvested and stained with 7aad. The frequency of dead targets in each culture condition was assayed by flow cytometry. Results are representative of two or more independent experiments. CD107a recruitment, IFN-γ and TNF-α production by CD3+ anti-Vβ2 and anti-CD19 CAR-Ts when cultured alone or in the presence of non-transduced JRT3-T3.5 cells (JRT), JRT3-T3.5 cells transduced with Vβ2+ TCR-GFP (Vβ+), or primary cells expressing Vβ2 (1YT) (see Figure 9C). Results are from a single experiment and are representative of two independent experiments. Enhanced killing of α-gal-loaded non-transduced and TCR Vβ1- or TCR Vβ2-GFP-transduced JRT3-T3.5 cells by anti-Vβ1 (see Figure 9D) or Vβ2 CAR-iNKTs, but not anti-Vβ1 or Vβ2 CAR-T cells (see Figure 9E). Where indicated, target cells were incubated with 200 ng / ml α-gal before coculture with effectors. Results shown are representative of two replicate experiments. The in vivo protocol used is shown in Figure 9F. 5 x 10 TCR Vβ JRT cells were suspended in Matrigel and injected subcutaneously. On day 18, 1 x 10 effector cells were injected intravenously into the tail vein. Challenge groups consisted of untreated (n = 5), Vβ2 CAR-iNKT (n = 7), or CD19 CAR-iNKT (n = 5). Tumor volume in each group was measured periodically using calipers (see Figure 9G). Tumors were excised and weighed at the end of the experiment (see Figure 9H). [Figure 10]Figure 10 shows ex vivo killing of adult T-cell leukemia / lymphoma (ATL) cells by anti-Vβ CAR-iNKT cells of the invention. Patient ATL01 had a malignant clone expressing TCR Vβ1, and ATL02 had a malignant clone expressing TCR Vβ2 (see Figure 10A). Ex vivo killing of CD4+CCR4+CD26- ATL cells expressing TCR Vβ1 (see Figure 10B) and Vβ2 (see Figure 10C) by anti-Vβ CAR-iNKT. Cryopreserved PBMCs from two ATL patients and a normal donor (ND) were stained with cell-trace violet and co-incubated in triplicate with anti-Vβ1, anti-Vβ2, or anti-CD19 CAR-iNKT at the indicated effector:target (PBMC) ratios. After 24 hours of coculture, cells were stained with viability dyes, anti-CD4, CCR4, CD26, TCR Vβ1 or 2, and TCR αβ, fixed, and the frequency of dead CD4+CCR4+CD26- T cells and CCR4-CD26± cells ("remainder of CD4+ T cells") was determined by flow cytometry. *In patient 2, there were insufficient numbers of cells to determine viability relative to CD4+ cells expressing other TCR Vβ subunits. [Figure 11]Figure 11 shows the effect of CAR-iNKT cells on the frequency of antiviral CTLs and CD4+ T cells expressing HTLV-1. The frequency of M158-66 and Tax11-19 HLA-A*0201 pentamer+ CD3+ T cells after coculture with CAR-iNKT (see Figures 11A and 11B). CD4-depleted PBMCs from three HLA-A*0201+ HTLV-1 carriers were stained with CellTraceViolet and cultured alone or with anti-Vβ1, anti-Vβ2, or anti-CD19 CAR-iNKT at a 1:1 ratio. After 16-18 hours of coculture, cells were stained with a viability dye, as well as anti-CD3, anti-CD8, and M158-66 or Tax11-19 pentamer. Cells were analyzed by flow cytometry to determine the frequency of viable pentamer+ CellTraceViolet+ CD3+ cells. The frequency of HTLV-1 Tax-expressing CD4+ cells after coculture with CAR-iNKT (see Figures 11C and 11D) is shown in Figures 11C and 11D. CellTraceViolet-stained positively selected CD4+ cells from the same donor were cultured alone or in the presence of Vβ2- or CD19-CAR iNKT. After 16–18 hours, cells were stained with viability dyes, anti-CD3, anti-CD4, anti-CD8, and anti-TCR Vβ2. Cells were then fixed and intracellularly stained with anti-Tax antibody. Cells were analyzed by flow cytometry to determine the frequency of live TCR Vβ2+ CD3+ cells and live Tax+ CD4+ cells. [Figure 12] Figure 12 shows the raw data from Figure 8c, i.e., the absolute frequency of CD3+ T cells expressing each TCR Vβ subunit expressed as a percentage of total CD3+ T cells. [Figure 13]Figure 13 shows the target cell line for CAR T cells of the invention. Primary T cells expressing the Vβ subunit of interest were enriched using an anti-TCRV β subunit antibody and magnetic beads (see Figure 13A). JRT3-T3.5 cells were transduced with a lentiviral expression vector encoding the TCR β chain and GFP (see Figure 13B). 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 relevant TCRRV β subunit and analyzed by flow cytometry. [Figure 14] Figure 14 shows the role played by iNKT iTCR cells of the invention in T cell killing. CD1d expression by in vitro expanded primary T cells and JRT3-T3.5 cells (JRT) (see Figure 14A). Cells were stained with viability dyes, anti-CD3 and anti-CD1d, and analyzed by flow cytometry. CD3 and CD1d expression by live cells is shown. Mouse weight over the course of the in vivo experiments described in Figures 9F-9H (see Figure 14B). [Figure 15] Figure 15 shows the effect of CAR-iNKT cells of the present invention on HTLV-1-expressing CD4+ T cells. The frequency of HTLV-1 Tax-expressing CD4+ cells after coculture with CAR-iNKT (see Figures 15A and 15B). Figure 15C shows the frequency of TCR Vβ1 and TCR Vβ2 CD3+ T cells after coculture. CellTraceViolet-stained positively selected CD4+ cells from the same donor were cultured alone or in the presence of Vβ1- or CD19-CAR iNKT. After 16-18 hours, cells were stained with viability dyes, anti-CD3, anti-CD4, anti-CD8, and anti-TCRVβ1. Cells were then fixed and intracellularly stained with anti-Tax antibody. Cells were then analyzed by flow cytometry to determine the frequency of live TCR Vβ1+ or TCR Vβ2+ CD3+ cells and live Tax+ CD4+ cells. [Figure 16]Figure 16 provides a comparison of the effect of linker size on CAR-T efficacy. Killing of in vitro-expanded autologous primary T cell lines by anti-TCR CAR-T cells and anti-CD19 CAR-T. Target cells were stained with CFSE and co-cultured in duplicate with effectors at a range of effector-to-target (E:T) ratios. After 24 hours, cells were harvested and stained with 7aad. The frequency of dead targets in each culture condition was assayed by flow cytometry. [Example]

[0226] As discussed below, we generated exemplary anti-TCR Vβ CAR constructs (referred to herein as clone BL37.2 for TCR Vβ1, MPB2D5 for TCR Vβ2, FIN9 for TCR Vβ9, and C21 for TCR Vβ11) (see Example 1) and tested their activity against select TCR Vβ-specifically expressing primary T cells and cancer T cell lines (see Example 2). We then generated effector T cells expressing selected anti-TCR Vβ CAR constructs, particularly iNKT cells expressing selected anti-TCR Vβ CAR constructs, and analyzed their suitability for immunotherapy of T-cell malignancies (see Example 3). The inventors then successfully demonstrated that anti-TCRVβ CAR-iNKT cells are highly active against ATL (see Example 4), do not impair CTL immunity to viral antigens, and do not promote HTLV-1 replicative activity, thereby exhibiting important safety characteristics required for the treatment of ATL and TCL.

[0227] Materials and Methods Ethics Statement Patients with ATL attended the National Centre for Human Retrovirology (Imperial College Healthcare NHS Trust, St Mary's Hospital, London), where they provided written informed consent. Research involving samples from patients with ATL was approved by the UK National Research Ethics Service (09 / H0606 / 106, 15 / SC / 0089, 20 / SC / 0226) and conducted under the supervision of the Communicable Diseases Research Group Tissue Bank.

[0228] Cell lines and chemicals HEK293T cells were passaged twice a week by trypsinization and maintained at 30–70% confluency in complete Dulbecco's modified Eagle's 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 cultured at 1 × 10 in complete RPMI medium containing 10% (v / v) FBS, 100 units / ml penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine, or in 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 ~1×10 6 C1R-CD1d cells (a gift from Vincenzo Cerundolo, Oxford University) were maintained at 1 × 10 cells / ml in complete RPMI. 5 ~1×10 6 cells / ml.

[0229] Generation of CAR constructs Four monoclonal antibodies were identified for proof-of-principle studies targeting TCR Vβ1 (clone BL37.2), Vβ2 (MPB2D5), Vβ9 (FIN9), and Vβ11 (C21), each purchased unconjugated 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 heavy and light chain fragments were amplified using isotype-specific primers. The amplified fragments were cloned into a standard cloning vector and transformed into Escherichia coli. Five colonies from 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 the constructs shown in FIGS.

[0230] Generation of lentivirus HEK293T cells were cultured at 4 × 10 in 10 ml of complete DMEM. 6 Cells were seeded at a density of 1000 μl / 10 cm plate. 24 hours later, the cells were transfected with the lentiviral expression vector and second-generation packaging plasmid using GeneJuice (Merck). 48 hours after transfection, the supernatant was collected, clarified by centrifugation at 500 g for 5 minutes, and then passed through a 0.45 μM filter. Lentiviral particles were concentrated by ultracentrifugation at 23,000 rpm for 2 hours at 4°C, resuspended in 200–300 μl of serum-free RPMI, and stored at −80°C until use. Virus titers were determined by titration using HEK-293T cells.

[0231] Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were purified from apheresis cones or EDTA-anticoagulated blood by density gradient centrifugation. Briefly, cells were collected from the cone and diluted to 100 ml with PBS. 25 ml of diluted cells were layered on 15 ml of histopaque (Sigma) and centrifuged at 800 g for 20 minutes. The buffy coat was collected, washed with PBS (400 g, room temperature for 5 minutes), and stored frozen in FBS containing 10% dimethyl sulfoxide (DMSO) until use.

[0232] Lentiviral transduction of T cells and iNKT cells PBMCs were rapidly thawed, washed twice in RPMI containing 10% FBS, and resuspended in a small volume of medium. iNKT cells were positively selected using magnetic bead separation according to the manufacturer's instructions (Miltenyi Biotech) by passing the cells through two consecutive LS columns. The purity of the positive fraction was assessed by flow cytometry staining using a fixable viability dye (Live / Dead Near-Infrared, Life Sciences) and anti-TCRVα24-Jα18 BV421 (clone 6B11), anti-TCRVB11 APC, anti-CD3 BV510, anti-CD4 BV605, and anti-CD8 FITC. For iNKT cultures, the positively selected fraction was cultured with irradiated (35 Gy) autologous feeder cells prepared from the negative fraction at a 1:1 ratio. For T cell cultures, the iNKT-depleted flow-through (flow cytometry) was used. The (through) fraction was cultured alone. T cells and iNKT cells were cultured in iNKT medium and stimulated with 50 ng / ml anti-CD3 and 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 retronectin-coated plates to obtain a multiplicity of infection (MOI) of 2.5–5 infectious units / cell. Cells were harvested, counted, added to transduction plates, and then centrifuged at 1000 g for 40 min at 32°C. On day 7 of iNKT culture, CD1d-expressing feeder cells were loaded with 200 ng / ml α-galactosylceramide (αGalCer, BioVision) for 2–4 h, irradiated, and added to the above cultures at a 1:1 ratio. Cells were fed twice weekly with fresh medium containing 150 IU / ml IL-15.

[0233] Quantification of lentiviral transduction efficiency Transduction efficiency was assessed starting 5 days after transduction. Cells were washed twice with cold PBS and incubated with 1 μg / ml streptavidin protein L (Pierce) for 45 minutes at 4°C. Cells were washed twice with PBS, stained with Live / dead near-infrared for 5 minutes at room temperature, washed with PBS 0.5% FBS, and stained with streptavidin-PE / BV421 and anti-CD3, anti-CD4, anti-CD8, anti-TCRVβ11, and anti-TCRVα24-Jα18 for 20 minutes at room temperature. Cells were acquired the same day using a BD Fortessa and analyzed using a Kaluza (Beckman Coulter) microscope.

[0234] Isolation and culture of primary T cell targets Autologous primary T cell lines expressing the desired TCR Vβ subunit were established by magnetic enrichment and in vitro expansion. PBMCs were stained with PE-conjugated anti-TCR Vβ antibody (Beckman Coulter) for 20 minutes at room temperature, washed once with complete RPMI, and incubated with anti-PE microbeads (Miltenyi Biotech) for an additional 20 minutes at 4°C. After one additional wash, 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.

[0235] Generation of GFP-TCR-expressing JRT3-T3.5 cells TCR β chain sequences containing the Vβ subunits of interest were identified in public databases. The table below summarizes the nomenclature of TCR genes (TRBV) and proteins (TCRVβ) and identifies proteins for which monoclonal antibodies are commercially available. Codon-optimized nucleotide sequences corresponding to the full-length beta chain were synthesized (using Genewiz) and cloned into a third-generation lentiviral expression vector (LeGO-iG2).

[0236] [Table 2(1)] [Table 2(2)]

[0237] 7aad cytotoxicity assay One day before cytotoxicity assessment, effector cells were counted and fed with fresh medium. Target cells were washed twice with PBS, incubated in 0.5 μM CFSE / Cell Trace Violet for 10 minutes at 37°C, washed twice in iNKT medium, and returned to culture with or without αGalCer as appropriate. On the day of the assay, cells were centrifuged and resuspended in iNKT medium containing 15 IU / ml IL-15. Cells were mixed to achieve a range of effector:target (E:T) ratios, plated in duplicate in a U-bottom 96-well plate, and centrifuged at 100 g for 1 minute. Cells were cultured for 4–24 hours, centrifuged at 800 g for 3 minutes, and resuspended in PBS containing 7aad (5 μg / ml). After a 20-minute incubation, each well was washed with 150 μl of PBS, centrifuged at 800 g for 3 minutes, and resuspended in 100 μl of PBS. In each well, the frequency of dead (7AAD+) target cells was counted by flow cytometry.

[0238] Cytotoxicity assay of fresh PBMCs One day before cytotoxicity assessment, effector cells were counted and fed with fresh medium. On the day of the assay, cryopreserved PBMCs were thawed, washed once with iNKT medium and once with PBS (400 g, 5 min), incubated in 0.5 μM CFSE / Cell trace violet for 10 min at 37°C, and washed twice with iNKT medium. PBMCs were plated in triplicate into U-bottom 96-well plates containing 15 IU / ml IL-15 with or without CAR-iNKT cells at a range of effector:target ratios. Plates were centrifuged at 100 g for 1 min and cultured for 24 h. Wells were washed with 150 μl of PBS (800 g, 3 min), stained with Live / Dead near-infrared for 5 min 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 for 20 min at room temperature. Cells were washed with 150 μl of FACS buffer, fixed with 150 μl of fixation buffer (Biolegend) for 20 min, washed once more with FACS buffer, and stored at 4°C until acquisition.

[0239] Intracellular cytokine staining and degranulation assay Effector cells were fed 24 hours (24 h) before assessing intracellular cytokine production / degranulation, and target cells were stained with CFSE. On the day of the experiment, target and effector cells were harvested, counted, and mixed to obtain a 1:1 E:T ratio in iNKT medium containing 20 μg / ml DNase, 15 IU / ml IL-15, 10.6 μM brefeldin, 2 μM monensin (1× protein transport inhibitor, eBioscience), and 2.5 μl of anti-CD107a-BV421 (clone H4A3) per 200 μl culture well. Cells were cultured for 6 h, centrifuged at 800 g for 3 min to remove the culture supernatant, and resuspended in live / dead NIR viability stain. After a 5-minute incubation at room temperature, 150 μl of PBS 0.5% FBS was added to each well, and the plate was washed again. Cells were resuspended in 150 μl of fixation / permeabilization buffer (eBiosciences FoxP3 Buffer Set, Life Technologies), incubated at room temperature for 30 min, and washed once with permeabilization buffer. Cells were stained with anti-CD3-BV510 (clone UCHT-1), anti-CD4-BV605 (RPA-T4), anti-CD8-AF700 (RPA-T8), anti-IFN-γ-BV711 (4S.B3), anti-TNF-α-PeCy7 (Mab11), anti-Granzyme B-PE (QA16A02), and perforin-APC (B-D48) diluted in permeabilization buffer. After a 30-min incubation at room temperature, cells were washed once with 150 μl of permeabilization buffer and resuspended in PBS 0.5% FBS until acquisition.

[0240] Pentamer staining HTLV-1-infected CD4+ cells express viral antigens in short-term ex vivo cultures and thus express HLA-A * Therefore, to minimize the possibility of downregulation of the cognate TCR on the CTLs due to antigen encounter during culture, CD4+ T cells were transfected with anti-CD4 PE and anti-PE microbeads to target HLA-A. *PBMCs from a 0201+ HTLV-1 carrier were depleted. 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 h of coculture, cells were stained with live / dead near-infrared as described above and resuspended in 40 μl of PBS containing 10 μl of HTLV-1 Tax11-19 or influenza A M158-66 pentamer-APC for 10 min, followed by the addition of anti-CD3-BV510 and anti-CD8-AF700. After an additional 20 min of incubation at room temperature, cells were washed and fixed for 30 min by resuspending in 150 μl of fixation buffer (Biolegend). After washing once with PBS, the frequency of live pentamer-positive, Cell Trace Violet+ CD8+ T cells in each culture was assessed by flow cytometry. Similarly, the CD4+ fraction was stained with live / dead anti-CD4-BV605 and anti-CD3-BV510, washed with PBS 7% NGS, and fixed with 150 μl of ebioscience FoxP3 fixation / permeabilization buffer for 30 minutes. Cells were then washed once in ebioscience permeabilization buffer and intracellularly stained with anti-Tax AF647 for 30 minutes. Cells were washed once more with 150 μl of permeabilization buffer, resuspended in PBS, and stored at 4°C in the dark until acquisition. The number of Tax+ CD4+ Cell Trace Violet+ cells was determined by flow cytometry.

[0241] In vivo experiments 5×10 6 TCR Vβ JRT cells were suspended in Matrigel and injected subcutaneously into the flanks of NSG mice. On day 18, 1 × 10 6 Effector cells were injected intravenously into the tail vein. Groups consisted of untreated (n=5), Vβ2 CAR-iNKT (n=7), or CD19 CAR-iNKT (n=5). Tumor volume in each group was measured periodically using calipers. At the end of the experiment, tumors were excised and weighed.

[0242] Off-target killing assay PBMCs from two normal donors were cultured alone or transduced with lentiviruses encoding CAR constructs specific for Vβ1, Vβ2, Vβ9, and Vβ11. Five days after transduction, transduction efficiency was assessed by protein L staining, and the frequency of CD3+ cells expressing each of the 24 TCR Vβ subunits was quantified. Values ​​were reported as the mean percentage of cells expressing each subunit, normalized to the frequency of cells expressing that subunit in untransduced PBMC controls from each individual.

[0243] result Example 1: Generation of CAR constructs targeting TCRVβ1, TCRVβ2, TCRVβ9, and TCRVβ11 We generated four lentiviral CAR constructs targeting TCR Vβ1 (expressed in 3.5% of CD3+ T cells in healthy donors), TCR Vβ2 (8.3%), TCR Vβ9 (3.2%), and TCR Vβ11 (1%). The anti-TCR Vβ1 CAR is referred to as the "BL37.2 CAR," the anti-TCR Vβ2 CAR as the "MPB2D5 CAR," the anti-TCR Vβ9 CAR as the "FIN9 CAR," and the anti-TCR Vβ11 CAR as the "C21 CAR."

[0244] Because hybridoma clones were not available for the first three mAbs, we determined the amino acid sequences of the mAbs by mass spectrometry and then employed reverse engineering of CARs. The corresponding nucleotide sequences were codon-optimized for expression in human cells and used to construct second-generation CARs (see Figures 1 and 2), in which the ectodomain contained the CD8α leader peptide-VL-(GGGGS)2 linker-VH-CD8α hinge-CD8α transmembrane domain, whereas the endodomain consisted of a portion of the CD8α cytoplasmic domain and the signaling (i.e., stimulatory and costimulatory) domains of CD28-CD3ζ.

[0245] 1 and 2, a schematic map of one embodiment of the coding sequence of the expression vector of the anti-TCRVβ CAR lentiviral CAR of the present invention is shown. It will be understood that the vector (DNA) also corresponds to the CAR (protein). The CAR comprises a human CD8α signal peptide, an antigen-binding domain comprising VL, 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.

[0246] To generate anti-TCRVβ11 CARs with codon-optimized VH / VL sequences, we used mRNA extracted from hybridoma C21 to amplify the expressed VH and VL chains. The resulting CAR constructs are shown in Figure 3. For each of the four CARs (i.e., "BL37.2 CAR," "MPB2D5 CAR," "FIN9 CAR," and "C21 CAR"), constructs with 2xG4S linkers and constructs with 3xG4S linkers were generated. These are illustrated. The position of each component in the constructs (5' to 3') follows the order disclosed in the schematic map in Figure 1.

[0247] The signal peptide is identical to that of human CD8α. Because the signal peptide matches the effector cell species (see below) and CD8α is highly expressed in T cells, the inventors believe that the signal peptide provides optimal expression of the CAR construct in effector cells. The hinge region of the CAR contains amino acids 128-210 of human CD8α, which includes 55 amino acids from the extracellular domain, the complete transmembrane helical domain, and 7 amino acids of the cytoplasmic domain. The intracellular domain of the CAR contains the CD3ζ stimulatory domain and the CD28 costimulatory domain.

[0248] Plasmids encoding lentiviral CAR constructs targeting TCRVβ1, TCRVβ2, TCRVβ9 and TCRVβ11 are shown in Figures 4, 5, 6 and 7, respectively.

[0249] Example 2: Evaluation of specificity, cytokine production, and cytotoxic activity of effector cells expressing anti-TCRVβ CAR constructs To begin testing the activity and specificity of the CARs described in Example 1, we transduced them into PBMC T cells from two healthy donors via lentiviral transduction. Five days after transduction, CAR expression in T cells was greater than 50% (Figures 8A and 8B). Simultaneously, we quantified the frequency of T cells expressing each of the 24 TCR Vβ chains in CAR-transduced PBMCs relative to untransduced T cells. We found that TCR Vβ1, Vβ2, Vβ9, and Vβ11-expressing T cells were almost completely depleted from PBMCs transduced with their respective cognate CAR constructs (median 98% reduction; range 90-100%) (Figures 8C and 12). In contrast, we observed a 4% increase in the median frequency of non-targeted Vβ subunits in transduced cultures compared to non-transduced controls, indicating a lack of "off-target" killing by TCR Vβ family-specific CAR-T cells.

[0250] In summary, anti-TCRVβ1, TCRVβ2, TCRVβ9 and TCRVβ11 CARs are robustly expressed, active and selective for their cognate TCRVβ chain targets.

[0251] Example 3: Anti-TCRV β-chain CAR against primary and cancer T cell lines To further explore the anti-T cell activity and TCR Vβ specificity of the four CAR constructs, we first tested TCR Vβ CAR-T cells against expanded autologous primary T cell lines highly purified to express the TCR Vβ chain of interest (Figure 13A). We found that all four anti-TCR Vβ CAR-T cells selectively killed their cognate T cell lines. However, the level of cytotoxicity varied, reflected by the intensity of staining with the corresponding mAbs, being both highest for anti-TCR Vβ1 and β2 and lowest for TCR Vβ9 (Figure 8D).

[0252] Next, we engineered the JRT3-T3.5 T cell line to express the TCR Vβ1 or 2 chain as a target. JRT3-T3.5 is a derivative of the Jurkat T cell line, which itself was derived from a patient with TCR Vβ lymphoblastic T cell lymphoma. Due to the deletion of the endogenous TCR Vβ chain gene, JRT3-T3.5 cells lacked expression of TCR and CD3. Therefore, as expected, introduction of exogenous TCR Vβ cDNA restored expression of both (Figure 13B). Accordingly, we found that anti-TCR Vβ1 and 2 CAR-T cells killed JRT3-T3.5 cells expressing their cognate target TCR Vβ, but not the parental cell line (Figure 8E).

[0253] In a complementary functional approach, we found that upon intracellular staining, anti-TCRVβ2 CAR-T cells expressed IFNg, TNFα, and CD107a (indicative of cytotoxic degranulation) when co-cultured with TCRVβ2-expressing JRT3-T3.5 cells or primary T cell lines, but not when cultured in the presence of parental JRT3-T3.5 or alone, and that more than 30% of CD4+ and CD8+ CAR-T cells were polyfunctional, i.e., co-expressing at least two molecules (Figures 8E and 8F).

[0254] Taken together, these findings demonstrate that the anti-TCRVβ CAR of the present invention, developed by reverse engineering, is highly specific and active in vitro.

[0255] Example 4: Anti-TCRVβ CAR-iNKT cells for "off-the-shelf" immunotherapy of T-cell malignancies Previous studies have demonstrated the feasibility of deploying iNKT cells as effector cells for CAR immunotherapy of hematologic cancers. Therefore, we generated anti-TCRVβ1, 2, 9, and 11 CAR-iNKT cells using our established manufacturing protocol. Anti-TCRVβ1, 2, and 9 CAR-iNKT cells efficiently killed JRT3-T3.5 and primary T cell lines expressing the corresponding TCR β chains (Figures 9A and 9B). However, CD4+ and CD4- CAR-iNKT cells specifically expressed or coexpressed IFNγ, TNFα, and CD107a upon stimulation with TCRVβ2-expressing JRT3-T3.5 and primary T cells (Figure 9C).

[0256] Because JRT3-T3.5 cells express CD1d (Fig. 14A), we hypothesized that anti-TCRVβ CAR-iNKT cell reactivity potently and selectively activates iNKT cells. 16、17 We tested whether iTCR activity could be enhanced in the presence of aGC, a glycolipid ligand for CD1d. We found that anti-TCRVβ1 and 2 CAR-T cytotoxicity against parental and cognate TCRVβ-expressing JRT3-T3.5 cells was not enhanced in the presence of aGC, whereas anti-TCRVβ1 and 2 CAR-iNKT cytotoxicity was enhanced against both targets, and more so against parental JRT3-T3.5 T cells (Figures 9D and 9E), thus demonstrating the functional relevance of iTCR in the anti-tumor activity of CAR-iNKT cells.

[0257] We further tested the activity of TCR Vβ CAR-iNKT cells in an in vivo model of T cell lymphoma in which TCR Vβ2-expressing JRT3-T3.5 T cells were injected subcutaneously into the flanks of NSG mice (Figure 9F). After tumor engraftment, mice were left untreated or injected with 10 T cells per mouse. 6JRT3-T3.5 T cells were treated with intravenous transfer of either TCR Vβ2 or CD19 CAR-iNKT cells. Consistent with the lack of CD19 expression by JRT3-T3.5 T cells, CD19 CAR-iNKT cells had no effect on T cell lymphoma tumor growth; in contrast, TCR Vβ2 CAR-iNKT cells significantly inhibited tumor volume and weight (Figures 9G and 9H).

[0258] Finally, CAR-iNKT cell-treated mice showed no evidence of excessive weight loss or other clinical signs of aGVHD after 42 days of monitoring (Figure 14B), thus supporting the idea of ​​using allogeneic iNKT cells as an "off-the-shelf" platform for cancer immunotherapy without the risk of aGVHD.

[0259] Collectively, these data demonstrate the therapeutic potential of the allogeneic TCR Vβ CAR-iNKT cells of the present invention for the treatment of T-cell lymphoma.

[0260] Example 5: Anti-TCRVβ CAR-iNKT cells are active against ATL Similar to the tests performed on JRT3-T3.5 T cell lymphoma cells, we also tested the reactivity of allogeneic anti-TCRVβ1 and 2 CAR-iNKT cells against PBMCs from normal donors and against PBMCs from two ATL patients enriched for lymphoma cells clonally expressing TCRβ1 or TCRβ2 (Figure 10A). As we previously demonstrated, in addition to their clonal TCRs, ATL malignant cells also coexpress CD4 and CCR4 and are negative for CD26 (Figure 10A). Therefore, we cocultured matched anti-TCRβ CAR-iNKT and anti-CD19 CAR-iNKT with PBMCs from each ATL and normal donor at a range of E:T ratios. As we previously reported, co-culture with anti-TCRVβ CAR-iNKT cells can block subsequent staining with the same anti-TCRVβ antibody clone, so we hypothesized that the immunophenotype was CD4+CCR4 med / hiWe used CD26- to identify lymphoma / leukemia cells in PBMCs from patients with ATL. We found that in patients 1 and 2, CCR4+CD26- cells comprised 90% of CD4+ cells, and nearly all expressed TCR Vβ1 or TCR Vβ2, respectively, whereas in normal donors, CCR4+CD26- cells comprised 25% of CD4+ cells, and TCR Vβ1 and TCR Vβ2 accounted for less than 8%.

[0261] At a 1:1 effector:CD4+ T cell ratio, anti-TCRVβ1 CAR-iNKT killed 75% of CD4+CCR4+CD26− cells and 10% of the "rest" (i.e., CCR4−CD26−) of CD4+ cells in patient 1. At the same ratios in patient 2, anti-TCRVβ2 CAR-iNKT killed 54% of CD4+CCR4+CD26− cells. The frequency of CD4+ cells expressing other TCRRVβ subunits was too low to determine whether there was off-target killing of normal CD4+ cells in this patient. When PBMCs from patients or normal donors were cocultured with anti-CD19 CAR-iNKT cells, minimal killing of CD4+CCR4+CD26− cells and "other CD4+" cells was observed. Similarly, minimal killing was observed after co-culture of PBMCs from normal donors with anti-TCRVβ1 and anti-TCRVβ2 CAR-iNKT cells, likely due to the low frequency of target cells present in the analyzed populations (Figures 10B and 10C).

[0262] In conclusion, anti-TCRVβ CAR-iNKT cells are highly active and specific for primary ATL cancer cells.

[0263] Example 6: Effect of anti-Vβ CAR-iNKT on antiviral CTL immunity and HTLV-1 viral status To further demonstrate selective targeting, we investigated the in vitro effects of our anti-TCRVβ CAR-iNKT cells on antiviral CD8+ T cells in HTVL-1-infected individuals.* PBMCs from O201+ HTLV-1 infected individuals (i.e., carriers) were cultured alone or in the presence of CAR-iNKT cells and subjected to HLA-A * The frequency of T cells binding to HTLV-1 Tax11-19 or influenza A M158-66 peptide-MHC pentamers was assessed (Figures 11A and 11B). When cultured alone, the frequency of influenza A M158-66 pentamer+ CD3+ T cells ranged from 0.14 to 0.23% of CD3+ cells, and the frequency of HTLV-1 Tax11-19 pentamer+ CD3+ T cells ranged from 0.23 to 0.88%. When cocultured with Vβ1-, Vβ2-, or CD19-CAR-iNKT cells, the frequency of pentamer+ cells was unchanged (Figures 11A and 11B). Efficient depletion of TCR Vβ1- and TCR Vβ2-expressing T cells by their cognate CAR-iNKT cells was confirmed in parallel cultures from the same donor (Figure 15C).

[0264] Because target cell killing by CAR-iNKT cells is associated with the secretion of proinflammatory cytokines, we wondered whether CAR-iNKT-mediated killing enhanced spontaneous expression of HTLV-1 Tax, 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 for 18 hours alone or in the presence of anti-Vβ1, anti-Vβ2, and anti-CD19 CAR-iNKT cells, and then assessed by intracellular staining for the frequency of Tax-expressing CD4+ T cells. Despite clear evidence of CAR-mediated depletion of T cells expressing the target TCR Vβ molecule, there was no change in the frequency of Tax+ CD4+ T cells in any of the CAR-iNKT cocultures compared with cells cultured alone (Figures 11C and 11D, 15A and 15B).

[0265] Therefore, CAR-iNKT cells targeting the TCR V β chain do not impair CTL immunity to viral antigens or promote HTLV-1 replication activity, both of which are important considerations for the safe treatment of TCL and ATL.

[0266] Example 7: Comparison of CAR constructs with (G4S)2 and (G4S)3 linkers To compare the effect of linker length on CAR efficiency in vitro, PBMC T cells were transduced with TCR Vβ1 and Vβ2 CARs containing either (G4S)2 or (G4S)3 linkers and co-cultured with primary T cell target cells expressing either TCR Vβ1 or TCR Vβ2.

[0267] Consideration The present invention demonstrates that anti-TCRVβ CAR immunotherapy can be developed as a highly sensitive, specific, and effective strategy for the treatment of TCRβ-expressing T cell malignancies. Given that the distribution of the TCRβ repertoire in T cell malignancies is similar to that in normal T cells, approximately 15% of TCRs can be targeted by the CARs developed and tested in this invention, thus paving the way for the development of CARs for all TCRβ families for which mAbs are available (currently approximately 70% of TCRβ chain families) or can be developed. Of the three exemplified TCRβ CARs, the best in vitro killing was observed against TCRβ1 and 2, with a lower degree of killing observed against TCRβ9-expressing targets. This pattern correlated with the intensity of T cell staining by the corresponding mAbs, suggesting low affinity of the anti-TCRVβ9 mAb, and therefore the corresponding CAR, and highlighting the need to select high-affinity mAbs.

[0268] Robust anti-TCL activity of anti-TCR Vβ CARs was demonstrated using two effector platforms. Thus, T cells and iNKT cells offer the promise of off-the-shelf immune cell therapies, either autologous or allogeneic, respectively. The subcutaneous TCL model used to test the in vivo efficacy of TCR Vβ CAR-iNKT cells is faithful to human TCL, where the skin is often the primary or secondary site of disease. While 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 lymphoma suggested a lack of significant toxicity and aGVHD. 30、31 The data also suggest that CAR-iNKT cells offer an additional therapeutic advantage over CAR-T cells in TCR Vβ and CD1d co-expressing cases of T-cell malignancies, such as T lymphoblastic lymphoma, as exemplified by the Jurkat T-cell line. Incorporation of αGalCer into the therapeutic approach could result in further enhancement of CAR-iNKT cell-mediated anti-leukemia / lymphoma effects.

[0269] Encouragingly, anti-TCR Vβ CAR-iNKT immunotherapy does not appear to affect adaptive antiviral immunity. There was no change in the frequency of influenza-specific or HTLV-1 Tax-specific CD8+ T cells when PBMCs from HTLV-1 carriers were cultured with or without anti-TCR Vβ CAR-iNKT. We also evaluated the effect of CAR-iNKT activity on proviral expression by by-standing HTLV-1-infected CD4+ T cells and observed no difference in HTLV-1 Tax expression in the presence or absence of CAR-iNKT cells. Thus, at least in short-term in vitro assays, we ruled out the possibility that bystander activation of HTLV-1-infected T cells favors HTLV-1 proviral reactivation.

[0270] overview In this disclosure, the inventors have successfully demonstrated that the novel and innovative anti-TCR Vβ CAR precision medicine approach for the treatment of TCL and ATL disclosed herein is applicable to all TCR Vβ chains. Due to their distinct advantages discussed above, the use of an allogeneic iNKT cell platform as effector cells is preferred, which will enable the rapid deployment of pre-manufactured allogeneic anti-TCR Vβ CAR-iNKT cells in HTLV-1 endemic regions and in combination with advanced clinical trial designs such as Bayesian optimal interval design. Such off-the-shelf anti-TCR Vβ CAR-iNKT cells are not only useful in patients with ATL, but also in patients with ATL, as we have recently shown, where clonal T cell expansion above a certain threshold predicted a high risk of progression to ATL, a highly incurable cancer. 32、33 , can also be tested in individuals infected with HTLV-1.

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Claims

1. A chimeric antigen receptor (CAR) construct specific for the T cell receptor (TCR) beta chain variable region (Vβ) subunit of a T cell.

2. 2. The CAR construct of claim 1, which is specific for pathogenic T cells.

3. 3. The CAR construct of claim 2, wherein the pathogenic T cell is either (i) a malignant pathogenic T cell, or (ii) a non-malignant pathogenic T cell.

4. 4. The CAR construct of any one of claims 1 to 3, wherein the Vβ subunit is selected from the group of Vβ subunits shown in Table 1.

5. The CAR construct of any one of claims 1 to 4, wherein the CAR construct targets a TCR Vβ subunit on a T cell selected from the group consisting of the following Vβ subunits: TCR-Vβ1, TCR-Vβ2, TCR-Vβ9, and TCR-Vβ11.

6. 6. The CAR construct of any one of claims 1 to 5, wherein the CAR construct is specific for TCR-Vβ1, and the TCR-Vβ1 subunit may comprise an amino acid sequence substantially as set forth in SEQ ID NO: 1, or a variant or fragment thereof.

7. 7. The CAR construct of any one of claims 1 to 6, wherein the CAR construct is specific for TCR-Vβ2, and the TCR-Vβ2 subunit may comprise an amino acid sequence substantially as set forth in SEQ ID NO: 2, or a variant or fragment thereof.

8. 8. The CAR construct of any one of claims 1 to 7, wherein the CAR construct is specific for TCR-Vβ9, and the TCR-Vβ9 subunit may comprise an amino acid sequence substantially as set forth in SEQ ID NO: 3, or a variant or fragment thereof.

9. 9. The CAR construct of any one of claims 1 to 8, wherein the CAR construct is specific for TCR-Vβ11, and the TCR-Vβ11 subunit may comprise an amino acid sequence substantially as set forth in SEQ ID NO: 4, or a variant or fragment thereof.

10. The CAR construct comprises: a) signaling peptides, b) an antigen-binding domain or antigen-binding portion specific for said TCR-Vβ subunit, preferably an anti-TCRVβ antibody or a functional fragment thereof specific for said 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 transmembrane domain and a cytoplasmic region; d) a primary stimulatory domain, and / or e) a costimulatory domain, preferably two or more costimulatory domains 10. The CAR construct of any one of claims 1 to 9, comprising:

11. 11. The CAR construct of any one of claims 1 to 10, wherein the CAR construct comprises a signaling peptide, wherein the signaling peptide comprises human CD8α, or a fragment or variant thereof.

12. 12. The CAR construct of claim 11, wherein the CAR construct comprises a signaling peptide comprising an amino acid sequence substantially as set forth in SEQ ID NO: 5, or a fragment or variant thereof, and / or the signaling peptide is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 6, or a fragment or variant thereof.

13. The CAR construct of any one of claims 1 to 12, wherein the CAR construct comprises an antigen-binding domain or antigen-binding portion specific for the TCR-Vβ subunit, the antigen-binding domain or antigen-binding portion comprising an anti-TCRVβ antibody or a functional fragment thereof specific for the TCR-Vβ subunit, and the antigen-binding domain specific for the TCR-Vβ subunit may comprise a single-chain variable fragment (scFv) domain of an anti-TCRVβ antibody.

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

15. the CAR construct comprises an scFv comprising a VL and / or a VH derived from an anti-TCRV β2 antibody; (i) the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 11, or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 12, or a fragment or variant thereof; and / or (ii) the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 13, or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 14, or a fragment or variant thereof.

15. A CAR construct according to any one of claims 1 to 14.

16. the CAR construct comprises an scFv comprising a VL and / or a VH derived from an anti-TCRVβ9 antibody; (i) the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 15, or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 16, or a fragment or variant thereof; and / or (ii) the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 17, or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 18, or a fragment or variant thereof.

16. A CAR construct according to any one of claims 1 to 15.

17. the CAR construct comprises an scFv comprising a VL and / or a VH derived from an anti-TCRVβ11 antibody; (i) the CAR construct comprises a VL chain having an amino acid sequence substantially as set forth in SEQ ID NO: 19, or a fragment or variant thereof, and / or the VL chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 20, or a fragment or variant thereof, and / or (ii) the CAR construct comprises a VH chain having an amino acid sequence substantially as set forth in SEQ ID NO: 21, or a fragment or variant thereof, and / or the VH chain is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 22, or a fragment or variant thereof.

17. A CAR construct according to any one of claims 1 to 16.

18. 18. The CAR construct of any one of claims 14 to 17, wherein the VH and VL sequences are separated by a linker sequence comprising at least one amino acid sequence substantially as set forth in SEQ ID NO: 23, or a fragment or variant thereof.

19. 19. The CAR construct of any one of claims 1 to 18, wherein the CAR construct comprises a hinge domain, the 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. (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 set forth in SEQ ID NO: 24, or a fragment or variant thereof; (iii) the hinge comprises or consists essentially of the amino acid sequence set forth 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 set forth in SEQ ID NO: 26, or a fragment or variant thereof.

20. The CAR construct of claim 19.

21. 21. The CAR construct of claim 20, wherein the hinge domain comprises or is derived from a human CD8α sequence comprising an amino acid sequence substantially as set forth in SEQ ID NO: 51, or a fragment or variant thereof, or a portion thereof.

22. (i) the hinge domain comprises the extracellular domain of human CD8α defined by amino acids 138-182 of human CD8α represented by 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 than amino acids 138-182 of human CD8α represented by SEQ ID NO:51; (iii) the hinge domain is at least 4, 5, or 6 amino acids longer at the N-terminus than amino acids 138-182 of human CD8α represented by 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 than amino acids 138-182 of human CD8α represented by SEQ ID NO:

51.

22. The CAR construct of claim 21.

23. (i) the hinge domain comprises the extracellular domain of human CD8α defined by amino acids 137-182, 136-182, or 135-182 of human CD8α represented by SEQ ID NO:51, or a portion thereof; (ii) the hinge domain comprises the extracellular domain of human CD8α defined by amino acids 134-182, 133-182, or 132-182 of human CD8α as represented by SEQ ID NO: 51, or a portion thereof; and / or (iii) the hinge domain comprises the extracellular domain of human CD8α defined by amino acids 131-182, 130-182, 129-182, or 128-182 of human CD8α represented by SEQ ID NO: 51, or a portion thereof.

23. The CAR construct of claim 22.

24. 22. The CAR construct of claim 21, wherein the hinge domain comprises the cytoplasmic domain of human CD8α defined by amino acids 204-206 of human CD8α represented by SEQ ID NO:51, or a portion thereof.

25. (i) the cytoplasmic domain is at least one amino acid longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO:51; (ii) the cytoplasmic domain is at least two amino acids longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO:51; (iii) the cytoplasmic domain is at least 3 amino acids longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO:51; and / or (iv) the cytoplasmic domain is at least four amino acids longer at the C-terminus than amino acids 204-206 of human CD8α represented by SEQ ID NO:

51.

25. The CAR construct of claim 24.

26. (i) the cytoplasmic domain is defined by amino acids 204-207 of human CD8α represented by SEQ ID NO:51; (ii) the cytoplasmic domain is defined by amino acids 204-208 of human CD8α represented by SEQ ID NO:51; (iii) the cytoplasmic domain is defined by amino acids 204 to 209 of human CD8α represented by SEQ ID NO: 51; and / or (iv) the cytoplasmic domain is defined by amino acids 204 to 210 of human CD8α represented by SEQ ID NO:

51.

26. The CAR construct of claim 25.

27. The CAR construct comprises a hinge domain, the hinge domain comprising: (i) a human CD8α extracellular domain having an amino acid sequence substantially as set forth in SEQ ID NO: 24, or a fragment or variant thereof, or a portion thereof; (ii) a human CD8α transmembrane (TM) domain having an amino acid sequence substantially as set forth in SEQ ID NO: 49, or a fragment or variant thereof, or a portion thereof; (iii) a human CD8α cytoplasmic domain having an amino acid sequence substantially as set forth in SEQ ID NO: 50, or a fragment or variant thereof, or a portion thereof; 20. The CAR construct of claim 19, comprising:

28. 28. The CAR construct of any one of claims 1 to 27, wherein the CAR construct comprises an intracellular domain, the intracellular domain comprising a primary stimulatory CD3ζ chain and a costimulatory domain of CD28.

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

30. 30. The CAR construct of claim 28 or claim 29, wherein the CAR construct comprises one or two costimulatory domains optionally selected from a CD28, 4-1BB signaling domain, and an OX40 signaling domain.

31. 31. The CAR construct of any one of claims 28 to 30, wherein the CAR construct comprises a stimulatory protein CD3ζ having an amino acid sequence substantially as set forth 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 set forth in SEQ ID NO: 30, or a fragment or variant thereof.

32. 32. The CAR construct of any one of claims 1 to 31, wherein the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 31 or SEQ ID NO: 33, or a fragment or variant thereof, and / or is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 32 or SEQ ID NO: 34, or a fragment or variant thereof.

33. 33. The CAR construct of any one of claims 1 to 32, wherein the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 35 or SEQ ID NO: 37, or a fragment or variant thereof, and / or is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 36 or SEQ ID NO: 38, or a fragment or variant thereof.

34. 34. The CAR construct of any one of claims 1 to 33, wherein the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 39 or SEQ ID NO: 41, or a fragment or variant thereof, and / or is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 40 or SEQ ID NO: 42, or a fragment or variant thereof.

35. 35. The CAR construct of any one of claims 1 to 34, wherein the CAR construct comprises an amino acid sequence substantially as set forth in SEQ ID NO: 43 or SEQ ID NO: 45, or a fragment or variant thereof, and / or is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 44 or SEQ ID NO: 46, or a fragment or variant thereof.

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

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

38. 38. An effector cell expressing a CAR construct of any one of claims 1 to 35, or comprising a nucleic acid of claim 36, or a vector of claim 37.

39. 39. The effector cell of claim 38, wherein the effector cell is (i) a normal or conventional αβ T cell, or (ii) an innate lymphoid cell, optionally an invariant natural killer T (iNKT) cell, a γδ T cell, or an NK cell.

40. 40. The effector cell of claim 38 or claim 39, wherein the effector cell is an iNKT cell.

41. 38. A method for producing an effector cell that expresses an anti-TCRV β CAR, the method comprising transducing an effector cell with the nucleic acid of claim 29 or the vector of claim 37 such that the effector cell expresses the anti-TCRV β CAR.

42. 42. The method of claim 41 , wherein the effector cell is (i) a normal or conventional αβ T cell, or (ii) an innate lymphocyte, preferably an invariant natural killer T (iNKT) cell, a γδ T cell, or an NK cell.

43. 43. The method of claim 41 or claim 42, wherein the method comprises an initial step of isolating the effector cells from peripheral blood cells (PBCs), and the effector cells are activated with one or both of a CD3 antibody and a CD28 antibody.

44. 44. The method of 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 described in any one of claims 1 to 35, a nucleic acid described in claim 29, a vector described in claim 37, or an effector cell described in any one of claims 38 to 40, and a pharmaceutically acceptable excipient.

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

47. 41. A CAR construct according to any one of claims 1 to 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 to 40 for use in therapy or diagnosis.

48. 41. The CAR construct of any one of claims 1 to 35, the nucleic acid of claim 36, the vector of claim 37, or the effector cell of any one of claims 38 to 40 for use (i) in immunotherapy, (ii) to treat, prevent or ameliorate cancer, (iii) to treat, prevent or ameliorate an autoimmune disease, or (iv) to treat, prevent or ameliorate any disease characterized by the presence of pathogenic T cells.

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

50. The T-cell malignancies include hematolymphoid 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-cell (TFH) cell lymphoma; peripheral T-cell lymphoma, NOS; EBV-positive NK cell and T-cell lymphoma; EBV 50. The CAR construct of any one of claims 1 to 35, the nucleic acid of claim 36, the vector of claim 37, or the effector cell of any one of claims 38 to 40 for use in any one of claims 47 to 49, wherein the CAR construct is selected from the group consisting of: positive T-cell lymphoproliferation; subcutaneous panniculitis-like lymphoma (SPTCL); angioimmunoblastic T-cell lymphoma (AITL); and cutaneous T-cell lymphoma (CTCL).

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

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

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