Binding domain

Abstract

The present invention provides variant antigen-binding domains that contain at least one mutation in the VH domain compared to a reference antibody and exhibit increased affinity for TRBC2 relative to the reference antibody. Also provided are antibodies, chimeric antigen receptors (CARs), and bispecific T cell engagers (BiTEs), cells comprising the CAR, and conjugates comprising the variant antigen-binding domain or the antibody. Additionally, the present invention provides medical uses, diagnostic methods, and personalized medicine methods using the products of the present invention.

Description

Field of the Invention

[0001] The present invention relates to variant antigen-binding domains that specifically bind to TRBC2. It also relates to cells and reagents that can be used for the treatment and diagnosis of T cell malignancies. Background of the Invention

[0003] Lymphoid malignancies can be broadly classified into those derived from T cells or B cells. T cell malignancies are a group of clinically and biologically heterogeneous disorders, collectively accounting for 10 - 20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histological subtypes are peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL). Among all acute lymphoblastic leukemias (ALL), approximately 20% are of T cell phenotype.

[0004] These conditions generally exhibit aggressiveness compared to, for example, B cell malignancies, where the estimated 5-year survival rate is only 30%. In the case of T cell lymphomas, they are associated with a high proportion of patients presenting with disseminated disease, adverse International Prognostic Index (IPI) scores, and an epidemic of extranodal disease. Chemotherapy alone is often ineffective, and less than 30% of patients are cured by current treatment methods.

[0005] Furthermore, unlike in B cell malignancies (where immunotherapies such as the anti-CD20 monoclonal antibody rituximab have significantly improved outcomes), there is currently no equally effective and minimally toxic immunotherapy available for the treatment of T cell malignancies. An important difficulty in developing immunotherapies for T cell disorders is the substantial overlap in marker expression between clonal T cells and normal T cells, and no single antigen can clearly distinguish clonal (malignant) cells.

[0006] Chimeric antigen receptor (CAR) T cells have shown promise in the treatment of refractory B cell malignancies. Targeting T cell malignancies may be equally effective, but the application of CARs in diseases such as T cell lymphoma has been hampered due to the lack of suitable target antigens. Unlike B cell lymphomas, where ablation of the B cell compartment is a tolerable toxicity and can be treated with intravenous immunoglobulin, disruption of the T cell compartment is poorly tolerated and leads to complications associated with the suppression of cell-mediated immunity.

[0007] Methods for treating T cell lymphoma and leukemia have been previously described in WO2015 / 132598, which include targeting the constant region of the TCRβ chain (TRBC). The method is based on the unique feature of the T cell receptor that each TCR encodes either TRBC1 or TRBC2 in a mutually exclusive manner. Since T cell lymphoma and leukemia are clonal populations of cells, each lymphoma will express on its surface a TCR that has either TRBC1 or TRBC2.

[0008] The monoclonal antibody Jovi-1 binds specifically to TRBC1 and has been used as a CAR binding domain for a therapy for treating T cell lymphoma (Maciocia et al., 2017, Nat Med 23:1416-23; WO2015 / 132598). This proposed therapy allows the treatment of a subset of patients expressing a TCR with a TRBC1 constant region.

[0009] To treat the entire patient population, a binder / CAR targeting TRBC2 is required. One way to obtain an antibody specific for TRBC2 is by phage selection against a human phage display library. Another approach lies in immunizing animals with a TRBC2-derived peptide and subsequently selecting specific antibodies. As disclosed in WO2015 / 132598, both methods have been successfully carried out and various TRBC2-specific binders have been generated.

[0010] The present invention provides alternative binders specific for TRBC2, which are potential therapeutic agents for treating TRBC2+ lymphoma or leukemia. Summary of the Invention

[0012] The inventors have solved the crystal structure of the TRBC1-specific monoclonal antibody JOVI-1 (Viney et al., 1992, Hybridoma 11:701-13) complexed with TRBC1-TCR to ( Figure 3 )). Based on this crystal structure, the original Jovi-1 antibody can be engineered to bind to TRBC2. This method is particularly attractive because many of the amino acids in the antibody form complementarity-determining regions that provide shape complementarity for TCR binding, while only a few amino acids are required for specificity for TRBC1. By computational biology and protein engineering, the inventors rationally designed a mutant form of the TRBC1 binder that is specific for TRBC2 and has reduced affinity for TRBC1.

[0013] Thus, in a first aspect, the present invention provides variant antigen-binding domains that comprise at least one mutation in the VH domain compared to a reference antibody having the following VH and VL domains: a VH domain having the sequence shown in SEQ ID NO:1 and a VL domain having the sequence shown in SEQ ID NO:2, wherein at least one mutation in the VH domain is selected from T28K, Y32K, and A100N, and wherein the variant antigen-binding domain shows increased affinity for TRBC2 relative to the reference antibody.

[0014] The variant antigen-binding domain may comprise at least two mutations in the VH domain selected from T28K, Y32K, and A100N. For example, it may comprise the mutations Y32K and A100N. The variant antigen-binding domain may also comprise the mutation T28R in the VH domain or alternatively the mutation G31K in the VL domain.

[0015] The variant antigen-binding domain may comprise the T28K, Y32K, and A100N mutations.

[0016] The variant antigen-binding domain may further comprise at least one mutation at positions selected from V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain, N35 in the VL domain, and R55 in the VL domain. The at least one additional mutation may be selected from:

[0017] a) In the VH domain:

[0018] - V2K, V2R,

[0019] - Y27F, Y27M, Y27N, Y27W,

[0020] - G31K, G31R, G31S,

[0021] - R98K,

[0022] - Y102F, Y102L,

[0023] - N103A, N103E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, N103Y,

[0024] - A107S,

[0025] and

[0026] b) In the VL domain:

[0027] - N35M, N35F, N35Y, N35K, N35R, and

[0028] -R55K.

[0029] The variant antigen-binding domain can be selected from variant antigen-binding domains comprising the following combinations of mutations:

[0030] -T28K, Y32F, A100N in the VH domain and N35K in the VL domain,

[0031] -T28K, Y32F, A100N in the VH domain,

[0032] -T28K, Y32F, A100N, Y27N in the VH domain,

[0033] -T28K, Y32F, A100N, G31K in the VH domain,

[0034] -T28K, Y32F, A100N, Y27M in the VH domain,

[0035] -T28K, Y32F, A100N, Y27W in the VH domain,

[0036] -T28K, Y32F, A100N in the VH domain and R55K in the VL domain,

[0037] -T28K, Y32F, A100N, N103H in the VH domain,

[0038] -T28K, Y32F, A100N, N103A in the VH domain,

[0039] -T28K, Y32F, A100N, N103Y in the VH domain,

[0040] -T28K, Y32F, A100N in the VH domain and N35R in the VL domain,

[0041] -T28K, Y32F, A100N, N103S and N35M in the VL domain in the VH domain,

[0042] -T28K, Y32F, A100N, N103M in the VH domain,

[0043] -T28K, Y32F, A100N, N103W and N35R in the VL domain in the VH domain,

[0044] -T28K, Y32F, A100N in the VH domain and N35F in the VL domain,

[0045] - T28K, Y32F, A100N, N103S in the VH domain and N35K in the VL domain,

[0046] - T28K, Y32F, A100N, R98K in the VH domain,

[0047] - T28K, Y32F, A100N, N103S in the VH domain and N35R in the VL domain,

[0048] - T28K, Y32F, A100N, N103L in the VH domain,

[0049] - T28K, Y32F, A100N, N103S in the VH domain and N35F in the VL domain,

[0050] - T28K, Y32F, A100N, N103S in the VH domain and N35Y in the VL domain,

[0051] - T28K, Y32F, A100N, N103L in the VH domain and N35M in the VL domain,

[0052] - T28K, Y32F, A100N, N103L in the VH domain and N35R in the VL domain,

[0053] - T28K, Y32F, A100N, N103W in the VH domain and N35K in the VL domain,

[0054] - T28K, Y32F, A100N, N103L in the VH domain and N35Y in the VL domain,

[0055] - T28K, Y32F, A100N, N103F in the VH domain,

[0056] - T28K, Y32F, A100N, N103W in the VH domain,

[0057] - T28K, Y32F, A100N, N103L in the VH domain and N35K in the VL domain,

[0058] - T28K, Y32F, A100N, N103L in the VH domain and N35F in the VL domain,

[0059] - T28K, Y32F, A100N, N103W in the VH domain and N35M in the VL domain,

[0060] -T28K, Y32F, A100N in the VH domain, and N35Y in the VL domain,

[0061] -T28K, Y32F, A100N, Y27F in the VH domain,

[0062] -T28K, Y32F, A100N, N103Q in the VH domain,

[0063] -T28K, Y32F, A100N, N103S in the VH domain,

[0064] -T28K, Y32F, A100N, N103M in the VH domain and N35F in the VL domain,

[0065] -T28K, Y32F, A100N, N103F in the VH domain and N35M in the VL domain,

[0066] -T28K, Y32F, A100N, N103F in the VH domain and N35F in the VL domain,

[0067] -T28K, Y32F, A100N, G31R in the VH domain,

[0068] -T28K, Y32F, A100N, N103W in the VH domain and N35F in the VL domain,

[0069] -T28K, Y32F, A100N, V2R in the VH domain,

[0070] -T28K, Y32F, A100N, G31S in the VH domain,

[0071] -T28K, Y32F, A100N, A107S in the VH domain,

[0072] -T28K, Y32F, A100N, N103E in the VH domain and N35M in the VL domain,

[0073] -T28K, Y32F, A100N, V2K in the VH domain,

[0074] -T28K, Y32F, A100N, N103E in the VH domain,

[0075] -T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35K in the VL domain,

[0076] - T28K, Y32F, A100N, Y102F in the VH domain, and N35F in the VL domain,

[0077] - T28K, Y32F, A100N, Y102F, N103M in the VH domain, and N35R in the VL domain,

[0078] - T28K, Y32F, A100N, Y102F in the VH domain, and N35R in the VL domain,

[0079] - T28K, Y32F, A100N, N103M in the VH domain, and N35M in the VL domain,

[0080] - T28K, Y32F, A100N, N103M in the VH domain, and N35Y in the VL domain,

[0081] - T28K, Y32F, A100N, N103M in the VH domain, and N35R in the VL domain,

[0082] - T28K, Y32F, A100N, N103F in the VH domain, and N35K in the VL domain,

[0083] - T28K, Y32F, A100N, Y102L, N103W in the VH domain, and N35R in the VL domain,

[0084] - T28K, Y32F, A100N, Y102L, N103W in the VH domain, and N35K in the VL domain,

[0085] - T28K, Y32F, A100N, Y102F, and,

[0086] - T28K, Y32F, A100N, Y102L, N103M in the VH domain, and N35R in the VL domain.

[0087] The variant antigen-binding domain may comprise the mutations T28K, Y32F, A100N in the VH domain and N35K in the VL domain.

[0088] The variant antigen-binding domain may comprise the mutations T28K, Y32F and A100N in the VH domain.

[0089] Compared with the reference antibody, the variant antigen-binding domain may further exhibit a reduced affinity for TRBC1.

[0090] The ratio of the affinity of the variant antigen-binding domain for TRBC2 and TRBC1 is at least 2.

[0091] The ratio of the affinity of the variant antigen-binding domain for TRBC2 and TRBC1 is at least 5.

[0092] The ratio of the affinity of the variant antigen-binding domain for TRBC2 and TRBC1 is at least 10.

[0093] The variant antigen-binding domain may further comprise an oligomerization domain.

[0094] In a second aspect, the present invention provides an antibody comprising the variant antigen-binding domain according to the first aspect of the present invention.

[0095] In a third aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the variant antigen-binding domain according to the first aspect of the present invention, a spacer, a transmembrane domain, and an intracellular domain.

[0096] The spacer may be selected from the human CD8 stem as shown in SEQ ID NO:7 and the COMP spacer as shown in SEQ ID NO:19.

[0097] In a fourth aspect, the present invention provides a bispecific T cell engager (BiTE) comprising the variant antigen-binding domain according to the first aspect of the present invention and a T cell activation domain.

[0098] In a fifth aspect, the present invention provides a nucleic acid sequence encoding the variant antigen-binding domain according to the first aspect of the present invention, the antibody according to the second aspect of the present invention, the CAR according to the third aspect of the present invention, or the BiTE according to the fourth aspect of the present invention.

[0099] In a sixth aspect, the present invention provides a vector comprising the nucleic acid sequence according to the fifth aspect of the present invention.

[0100] In a seventh aspect, the present invention provides a cell comprising the CAR according to the third aspect of the present invention.

[0101] In an eighth aspect, the present invention provides a method for preparing the cell according to the seventh aspect of the present invention, which comprises the step of transducing or transfecting a cell with the vector according to the sixth aspect of the present invention comprising a nucleic acid sequence encoding the CAR.

[0102] In a ninth aspect, the present invention provides a conjugate comprising the variant antigen-binding domain according to the first aspect of the present invention, or the antibody according to the second aspect of the present invention, and a detectable entity or a chemotherapeutic entity.

[0103] The conjugate may comprise a chemotherapeutic entity.

[0104] In a tenth aspect, the present invention provides a method for treating T cell lymphoma or leukemia in a subject, which comprises the step of administering to the subject a cell according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention, wherein the malignant T cells express TRBC2.

[0105] The T cell lymphoma or leukemia may be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0106] In an eleventh aspect, the present invention provides a cell according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention, for use in a medicament.

[0107] In a twelfth aspect, the present invention provides a cell according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention, for use in the treatment of T cell lymphoma or leukemia, wherein the malignant T cells express TRBC2.

[0108] The T cell lymphoma or leukemia may be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0109] In a thirteenth aspect, the present invention provides the use of a cell according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention in the preparation of a medicament for the treatment of T cell lymphoma or leukemia, wherein the malignant T cells express TRBC2.

[0110] T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0111] In a fourteenth aspect, the present invention provides a diagnostic agent comprising a variant antigen-binding domain according to the first aspect of the present invention or an antibody according to the second aspect of the present invention.

[0112] The diagnostic agent can be used for diagnosing T cell lymphoma or leukemia.

[0113] T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0114] In a fifteenth aspect, the present invention provides a method for diagnosing T cell lymphoma or leukemia in a subject, which comprises the step of contacting a variant antigen-binding domain according to the first aspect of the present invention or an antibody according to the second aspect of the present invention with a sample containing T cells from the subject.

[0115] The method for diagnosing T cell lymphoma or leukemia in a subject further comprises the step of determining the percentage of TRBC2-positive T cells in the sample.

[0116] A percentage of 70% or higher of TRBC2-positive T cells in the sample can indicate the presence of T cell lymphoma or leukemia.

[0117] The sample can be a blood sample or can be derived from a blood sample.

[0118] T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0119] In a sixteenth aspect, the present invention provides a method for identifying a subject having T cell lymphoma or leukemia suitable for treatment with the cells according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention, comprising determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0120] If the percentage of TRBC2-positive T cells in the sample is 70% or higher, the subject is suitable for treatment with the cells according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention.

[0121] The sample can be a blood sample or can be derived from a blood sample.

[0122] The T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0123] In a seventeenth aspect, the present invention provides a method for selecting a therapy comprising the cells according to the seventh aspect of the present invention, or an antibody according to the second aspect of the present invention, or a BiTE according to the fourth aspect of the present invention, or a conjugate according to the ninth aspect of the present invention for treating a subject, which comprises determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0124] If the percentage of TRBC2-positive T cells in the sample is 70% or higher, the therapy is selected for treating the subject.

[0125] The sample can be or can be derived from a blood sample.

[0126] The T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 .Schematic diagram of the αβ T cell receptor / CD3 complex. The T cell receptor is formed by six different protein chains, which must be assembled in the endoplasmic reticulum for cell surface expression. Four proteins of the CD3 complex (CD3z, CD3γ, CD3ε, and CD3δ) cover the T cell receptor (TCR). This TCR confers specificity for a particular antigen to the complex and consists of two chains: TCRα and TCRβ. Each TCR chain has a variable component on the distal side of the membrane and a constant component on the proximal side of the membrane. Almost all T cell lymphomas and many T cell leukemias express the TCR / CD3 complex.

[0128] Figure 2 : Separation of the T cell receptor β constant regions (TRBC)-1 and TRBC2 during T cell receptor rearrangement. Each TCRβ chain is formed by genomic recombination of a specific β variable region (V), diversity region (D), joining region (J), and constant region (TRBC). The human genome contains two very similar and functionally equivalent TRBC loci, called TRBC1 and TRBC2. During TCR gene rearrangement, the J region recombines with either TRBC1 or TRBC2. This rearrangement is permanent. T cells express many copies of a single TCR on their surface, so each T cell will express a TCR whose β chain constant region is encoded by either TRBC1 or TRBC2.

[0129] Figure 3 .Structural interface between the Fab fragment of the TCRβ- and TRBC1-specific antibody Jovi-1.

[0130] Figure 4 .Structural diagram of TRBC1- and TRBC2-specific chimeric antigen receptors (CARs).

[0131] Figure 5 .Functional characterization of the anti-TRBC2 CAR.

[0132] IFN-γ production upon incubation of (A) anti-TRBC2 triple mutant CAR and (B) anti-TRBC1 CAR in the presence of TRBC1 or TRBC2.

[0133] Figure 6 .Cytotoxic activity of (A) anti-TRBC2 triple mutant CAR-T cells and (B) anti-TRBC1 CAR-T cells co-incubated with Raji WT, Raji TRBC1 + or Raji TRBC2 + cells.

[0134] Figure 7Antigen-specific activation of Jurkat cells transduced with anti-TRBC2 CAR and co-incubated with HPB TRBC1 and HPB TRBC2 cells. Jurkat cells (TRBC1+) were transduced with a second-generation anti-TRBC2 CAR construct and co-incubated with HPB TRBC1 and HPB TRBC2 cells at an E:T ratio of 1:1. Controls included untransduced Jurkat cells (NT), HPB-ALL cells with knockout of the TCR (referred to as HPB KO), and transduced Jurkat cells plated alone as a negative control, or plated with αCD3 / αCD28 antibodies as a positive assay control. N35K: conjugate with T28K, Y32F, A100N mutations in the VH domain of hJovi-1 and N35K in the VL domain; N103L: conjugate with T28K, Y32F, A100N, N103L mutations in the VH domain of hJovi-1; N103M-N35Y: conjugate with T28K, Y32F, A100N, N103M mutations in the VH domain of hJovi-1 and N35Y in the VL domain; Y102F-N103M-N35R: conjugate with T28K, Y32F, A100N, Y102F, N103M mutations in the VH domain of hJovi-1 and N35R in the VL domain; and Y102L-N103M-N35R: conjugate with T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain of hJovi-1 and N35R in the VL domain.

[0135] Figure 8. Cytotoxic activity of anti-TRBC2 CAR-T cells co-incubated with TRBC1+ HPB-ALL and TRBC2+ HPB-ALL cells. Peripheral blood mononuclear cells (PBMCs) were transduced with a second-generation anti-TRBC2 CAR construct. The transduced PBMCs were co-incubated with TRBC1+ HPB-ALL and TRBC2+ HPB-ALL cells at an E:T ratio of 1:2. Controls included untransduced cells (NT), cells transduced with anti-TRBC1 hJovi-1 CAR (JOVI), and HPB-ALL cells with knocked-out TCR (HPB-KO). N35K: conjugate with T28K, Y32F, A100N mutations in the VH domain of hJovi-1 and N35K in the VL domain; N103L: conjugate with T28K, Y32F, A100N, N103L mutations in the VH domain of hJovi-1; N103M-N35Y: conjugate with T28K, Y32F, A100N, N103M mutations in the VH domain of hJovi-1 and N35Y in the VL domain; Y102F-N103M-N35R: conjugate with T28K, Y32F, A100N, Y102F, N103M mutations in the VH domain of hJovi-1 and N35R in the VL domain; and Y102L-N103M-N35R: conjugate with T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain of hJovi-1 and N35R in the VL domain.

[0136] Figure 9 : Effects of different antibody forms on anti-TRBC2 antibody binding were analyzed by surface plasmon resonance (SPR). Detailed Description of the Invention

[0138] The inventors have solved the crystal structure of Jovi-1, which has been used to identify two key residues essential for TRBC1 specificity ( Figure 1 ). Interestingly, these residues are located in CDR 1 rather than CDR 3, while CDR 3 usually drives antibody specificity. Additionally, other residues very close to the TRBC1 epitope may be essential for engineering specificity from TRBC1 to TRBC2. Residues in Jovi-1 involved in TRBC1 binding or important for generating TRBC2 specificity are described herein.

[0139] The present invention provides variants of the antigen-binding domain of JOVI-1 that have increased affinity for the TCRβ constant region 2 (TRBC2) compared to JOVI-1.

[0140] 1. TCRβ Constant Region (TRBC)

[0141] T cell receptors (TCRs) are expressed on the surface of T lymphocytes and are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. When a TCR engages an antigen peptide and MHC (peptide / MHC), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and transcription factors that are either activated or released.

[0142] TCRs are disulfide-linked, membrane-anchored heterodimers, typically composed of highly variable alpha (α) and beta (β) chains, expressed as part of a complex with invariant CD3 chain molecules. T cells expressing this receptor are called α:β (or αβ) T cells (about 95% of total T cells). A minority of T cells express an alternative receptor formed by variable gamma (γ) and delta (δ) chains and are called γδ T cells (about 5% of total T cells).

[0143] Each α and β chain consists of two extracellular domains: a variable (V) region and a constant (C) region, which together form an antiparallel β-sheet of two immunoglobulin superfamily (IgSF) domains. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. The constant region of the TCR consists of short linker sequences in which cysteine residues form disulfide bonds that make the connection between the two chains.

[0144] The variable domains of both the TCR α and β chains have three hypervariable or complementarity-determining regions (CDRs). The variable region of the β chain also has an additional hypervariable region (HV4), however, this region generally does not contact the antigen and thus is not considered a CDR.

[0145] TCRs also contain up to five invariant chains γ, δ, ε (collectively called CD3) and ζ. The CD3 and ζ subunits mediate TCR signaling through specific cytoplasmic domains that interact with second messengers and adaptor molecules after antigen recognition by αβ or γδ. The cell surface expression of the TCR complex is preceded by the paired assembly of the subunits, in which both the transmembrane and extracellular domains of TCR α and β as well as CD3 γ and δ play a role.

[0146] Thus, a TCR typically consists of the CD3 complex as well as the TCR α and β chains, which in turn consist of variable and constant regions ( Figure 1 ).

[0147] The locus (Chr7:q34) that supplies the TCR β constant region (TRBC) has been duplicated in evolutionary history to give rise to two nearly identical and functionally equivalent genes: TRBC1 and TRBC2 ( Figure 2)。Each TCR will contain either TRBC1 or TRBC2 in a mutually exclusive manner, and thus each αβ T cell will express either TRBC1 or TRBC2 in a mutually exclusive manner.

[0148] The inventors have previously determined that, despite the similarity between the sequences of TRBC1 and TRBC2, it is possible to distinguish between them. The inventors have also previously determined that the amino acid sequences of TRBC1 and TRBC2 can be distinguished in situ on the surface of cells such as T cells (WO2015 / 132598).

[0149] 2. Variant Antigen-Binding Domain

[0150] In a first aspect, the present invention provides a variant antigen-binding domain, hereinafter referred to as " The variant antigen-binding domain of the present invention ", which contains at least one mutation in the VH domain compared to a reference antibody having the following VH and VL domains: a VH domain having the sequence shown in SEQ ID NO:1 and a VL domain having the sequence shown in SEQ ID NO:2, wherein at least one mutation in the VH domain is selected from T28K, Y32F, and A100N, and wherein the variant antigen-binding domain shows an increased affinity for TRBC2 relative to the reference antibody.

[0151] As used herein, the term " Variant " or " Mutant " refers to a polypeptide that is different from a specifically recited polypeptide (i.e., the reference or parental polypeptide) by amino acid insertions, deletions, and / or substitutions, produced using, for example, recombinant DNA technology or de novo synthesis. Variants and mutants are used interchangeably in the context of the present invention. The variant antigen-binding domains of the present invention include antigen-binding molecules in which one or several amino acid residues are modified by substitution, addition, and / or deletion such that the antigen-binding affinity of the reference or parental antigen-binding domain is substantially affected.

[0152] As used herein, the term " Antigen-binding domain" refers to the variable regions of each pair of light and heavy chains of an antibody, namely the VL and VH domains that form its binding site. They are characterized by the same general structure in which relatively conserved regions called frameworks (FRs) are linked to three hypervariable regions called complementarity-determining regions (CDRs) (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.; Chothia & Lesk, 1987, J Mol Biol 196:901-17). As used herein, the term "complementarity-determining region" or "CDR" refers to the region within an antibody that is complementary to the shape of an antigen. Thus, the CDRs determine the affinity (roughly the binding strength) and specificity of the protein for a particular antigen. The CDRs of each pair of two chains are arranged through the framework regions, thereby obtaining the function of binding to a specific epitope.

[0153] Compared to a reference antibody, the variant antigen-binding domain of the present invention comprises at least one mutation in the VH domain. As used herein, the term " Reference antibody " refers to the humanized JOVI-1 antibody, i.e., hJOVI-1, which comprises a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2. Murine JOVI-1 has been described by Viney et al. (1992; supra) and is commercially available (Abcam, ab5465). It has previously been determined that JOVI-1 is capable of differentiating cells based on the specific expression of TRBC1 or TRBC2 by specifically binding only to TRBC1 (WO 2015 / 132598).

[0154] SEQ ID NO:1 (VH domain of hJOVI-1):

[0155] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSS

[0156] SEQ ID NO:2 (VL domain of hJOVI-1):

[0157] DIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIK

[0158] Unless otherwise indicated herein, any reference to a VH domain refers to the VH domain of hJOVI-1 shown in SEQ ID NO: 1; and any reference to a VL domain refers to the VL domain of hJOVI-1 shown in SEQ ID NO: 2, unless otherwise indicated.

[0159] The inventors have determined that the presence of at least one mutation selected from T28K, Y32F, and A100N in the VH domain of the reference antibody triggers an increase in the affinity of the variant antigen-binding domain of the present invention for TRBC2 relative to the reference antibody (Example 1).

[0160] As used herein, the term " Affinity " refers to the strength of the interaction between the antigen-binding site of an antibody and an epitope. Compared to an antibody with low affinity, a high-affinity antibody will bind more antigen in a shorter period of time. Affinity is typically measured by the equilibrium dissociation constant (K D ) between the antibody and the antigen, which is the ratio of k off / k on . The affinity of an antigen-binding domain for any given antigen can be quantified using any conventional method, including but not limited to label-dependent methods such as direct and indirect ELISA and radioimmunoassay methods, and label-free methods capable of directly detecting and measuring the interaction in real time, such as surface plasmon resonance and biolayer interferometry.

[0161] The variant antigen-binding domain of the present invention has an increased affinity for TRBC2 compared to the reference antibody. The affinity of the variant antigen-binding domain for TRBC2 can be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, or at least 10,000% compared to the affinity of the reference antibody for TRBC2.

[0162] Variant antigen-binding domains of the invention may comprise the T28K mutation in the VH domain. Variant antigen-binding domains of the invention may comprise the Y32F mutation in the VH domain. Variant antigen-binding domains of the invention may comprise the A100N mutation in the VH domain.

[0163] Variant antigen-binding domains of the invention may comprise at least two mutations in the VH domain selected from T28K, Y32F, and A100N. The at least two mutations may be Y32F and A100N. In an embodiment, variant antigen-binding domains of the invention may comprise the mutations Y32F and A100N in the VH domain and further comprise the mutation T28R. In an embodiment, variant antigen-binding domains of the invention may comprise the mutations Y32F and A100N in the VH domain and further comprise the mutation G31R.

[0164] Variant antigen-binding domains of the invention may comprise the mutations T28K, Y32F, and A100N in the VH domain.

[0165] In another embodiment, variant antigen-binding domains of the invention comprise the mutations T28K, Y32F, and A100N in the VH domain and further comprise at least one mutation at a position selected from V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain and N35 and R55 in the VL domain. Variant antigen-binding domains of the invention may comprise at least one, two, three, four, five, six, or seven mutations at positions selected from V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain and N35 and R55 in the VL domain.

[0166] Variant antigen-binding domains of the invention may comprise the mutations T28K, Y32F, and A100N in the VH domain and may further comprise a mutation at position V2 in the VH domain, a mutation at position Y27 in the VH domain, a mutation at position G31 in the VH domain, a mutation at position R98 in the VH domain, a mutation at position Y102 in the VH domain, a mutation at position N103 in the VH domain, a mutation at position A107 in the VH domain, a mutation at position N35 in the VL domain, and a mutation at position R55 in the VL domain.

[0167] Variant antigen-binding domains of the invention may comprise the mutations T28K, Y32F, and A100N in the VH domain and further comprise at least one mutation selected from:

[0168] a) in the VH domain:

[0169] - V2K, V2R,

[0170] -Y27F, Y27M, Y27N, Y27W,

[0171] -G31K, G31R, G31S,

[0172] -R98K,

[0173] -Y102F, Y102L,

[0174] -N103A, N103E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, N103Y, and

[0175] -A107S,

[0176] and

[0177] b) in the VL domain:

[0178] -N35M, N35F, N35Y, N35K, N35R, and

[0179] -R55K.

[0180] The variant antigen-binding domains of the present invention may be selected from variant antigen-binding domains comprising the following combinations of mutations:

[0181] -T28K, Y32F, A100N, Y27N in the VH domain,

[0182] -T28K, Y32F, A100N, G31K in the VH domain,

[0183] -T28K, Y32F, A100N, Y27M in the VH domain,

[0184] -T28K, Y32F, A100N, Y27W in the VH domain,

[0185] -T28K, Y32F, A100N in the VH domain,

[0186] -T28K, Y32F, A100N in the VH domain and R55K in the VL domain,

[0187] -T28K, Y32F, A100N in the VH domain and N35K in the VL domain,

[0188] -T28K, Y32F, A100N, N103H in the VH domain,

[0189] -T28K, Y32F, A100N, N103A in the VH domain,

[0190] - T28K, Y32F, A100N, N103Y in the VH domain,

[0191] - T28K, Y32F, A100N in the VH domain and N35R in the VL domain,

[0192] - T28K, Y32F, A100N, N103S in the VH domain and N35M in the VL domain,

[0193] - T28K, Y32F, A100N, N103M in the VH domain,

[0194] - T28K, Y32F, A100N, N103W in the VH domain and N35R in the VL domain,

[0195] - T28K, Y32F, A100N in the VH domain and N35F in the VL domain,

[0196] - T28K, Y32F, A100N, N103S in the VH domain and N35K in the VL domain,

[0197] - T28K, Y32F, A100N, R98K in the VH domain,

[0198] - T28K, Y32F, A100N, N103S in the VH domain and N35R in the VL domain,

[0199] - T28K, Y32F, A100N, N103L in the VH domain,

[0200] - T28K, Y32F, A100N, N103S in the VH domain and N35F in the VL domain,

[0201] - T28K, Y32F, A100N, N103S in the VH domain and N35Y in the VL domain,

[0202] - T28K, Y32F, A100N, N103L in the VH domain and N35M in the VL domain,

[0203] - T28K, Y32F, A100N, N103L in the VH domain and N35R in the VL domain,

[0204] - T28K, Y32F, A100N, N103W in the VH domain and N35K in the VL domain,

[0205] - T28K, Y32F, A100N, N103L in the VH domain and N35Y in the VL domain,

[0206] - T28K, Y32F, A100N, N103F in the VH domain,

[0207] - T28K, Y32F, A100N, N103W in the VH domain,

[0208] - T28K, Y32F, A100N, N103L in the VH domain and N35K in the VL domain,

[0209] - T28K, Y32F, A100N, N103L in the VH domain and N35F in the VL domain,

[0210] - T28K, Y32F, A100N, N103W in the VH domain and N35M in the VL domain,

[0211] - T28K, Y32F, A100N, N103F in the VH domain and N35Y in the VL domain,

[0212] - T28K, Y32F, A100N, Y27F in the VH domain,

[0213] - T28K, Y32F, A100N, N103Q in the VH domain,

[0214] - T28K, Y32F, A100N, N103S in the VH domain,

[0215] - T28K, Y32F, A100N, N103M in the VH domain and N35F in the VL domain,

[0216] - T28K, Y32F, A100N, N103F in the VH domain and N35M in the VL domain,

[0217] - T28K, Y32F, A100N, N103F in the VH domain and N35F in the VL domain,

[0218] - T28K, Y32F, A100N, G31R in the VH domain,

[0219] - T28K, Y32F, A100N, N103W in the VH domain and N35F in the VL domain,

[0220] - T28K, Y32F, A100N, V2R in the VH domain,

[0221] - T28K, Y32F, A100N, G31S in the VH domain,

[0222] - T28K, Y32F, A100N, A107S in the VH domain,

[0223] - T28K, Y32F, A100N, N103E in the VH domain and N35M in the VL domain,

[0224] - T28K, Y32F, A100N, V2K in the VH domain,

[0225] - T28K, Y32F, A100N, N103E in the VH domain,

[0226] - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35K in the VL domain,

[0227] - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35F in the VL domain,

[0228] - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35R in the VL domain,

[0229] - T28K, Y32F, A100N, Y102F in the VH domain and N35R in the VL domain,

[0230] - T28K, Y32F, A100N, N103M in the VH domain and N35M in the VL domain,

[0231] - T28K, Y32F, A100N, N103M in the VH domain and N35Y in the VL domain,

[0232] - T28K, Y32F, A100N, N103M in the VH domain and N35R in the VL domain,

[0233] - T28K, Y32F, A100N, N103F in the VH domain and N35K in the VL domain,

[0234] - T28K, Y32F, A100N, Y102L, N103W in the VH domain and N35R in the VL domain,

[0235] - T28K, Y32F, A100N, Y102L, N103W in the VH domain and N35K in the VL domain,

[0236] - T28K, Y32F, A100N, Y102F in the -VH domain, and,

[0237] - T28K, Y32F, A100N, Y102L in the -VH domain and N35R in the VL domain.

[0238] These specific combinatorial mutations have been shown to alter binding to TRBC2 and TRBC1 in a manner useful for targeting TRBC2 (see Table 1).

[0239] Table 1: Affinity of variant antigen-binding domains of the invention for TRBC2 and TRBC1.

[0240]

[0241]

[0242] N / A: Not obtained; binding to TRBC1 is effectively zero and beyond the detection limit of the device.

[0243] In certain embodiments, the variant antigen-binding domain of the invention comprises the T28K, Y32F, and A100N mutations in the VH domain.

[0244] In another particular embodiment, the variant antigen-binding domain of the invention comprises the T28K, Y32F, A100N, and Y27N mutations in the VH domain.

[0245] In another particular embodiment, the variant antigen-binding domain of the invention can comprise the T28K, Y32F, A100N, and N103M mutations in the VH domain.

[0246] In another particular embodiment, the variant antigen-binding domain of the invention comprises the T28K, Y32F, A100N mutations in the VH domain and the N35K mutation in the VL domain.

[0247] In another particular embodiment, the variant antigen-binding domain of the invention comprises the T28K, Y32F, A100N, and N103L mutations in the VH domain.

[0248] In another particular embodiment, the variant antigen-binding domain of the invention comprises the T28K, Y32F, A100N, N103M mutations in the VH domain and the N35Y mutation in the VL domain.

[0249] In another specific embodiment, the variant antigen-binding domain of the present invention comprises the mutations T28K, Y32F, A100N, Y102F, N103M in the VH domain and the mutation N35R in the VL domain.

[0250] In another specific embodiment, the variant antigen-binding domain of the present invention comprises the mutations T28K, Y32F, A100N, Y102L, N103M in the VH domain and the mutation N35R in the VL domain.

[0251] Advantageously, the variant antigen-binding domain of the present invention not only shows an increased affinity for TRBC2 relative to the reference antibody, but also shows a decreased affinity for TRBC1 compared to the reference antibody. This change in antigen specificity will allow the variant antigen-binding domain to distinguish TRBC2 and TRBC1 by showing preferential binding to TRBC2. Thus, in another embodiment, the variant antigen-binding domain further shows a decreased affinity for TRBC1 compared to the reference antibody.

[0252] The variant antigen-binding domain of the present invention has a decreased affinity for TRBC1 compared to the reference antibody. The affinity of the variant antigen-binding domain for TRBC2 can be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000% or at least 10,000% compared to the affinity of the reference antibody for TRBC1.

[0253] The ratio of the affinity of the variant antigen-binding domain of the present invention for TRBC2 to its affinity for TRBC1 can be at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 15, or at least 20, or at least 25, or at least 50, or at least 100, or at least 500, or at least 1,000 or greater.

[0254] By increasing the affinity of the variant antigen-binding domain of the present invention in Example 6, the inventors unexpectedly found that not only did the affinity of the variant antigen-binding domain of the present invention for TRBC2 increase, but its low affinity for TRBC1 was maintained. Thus, the specificity of the variant antigen-binding domain of the present invention for TRBC2 was significantly improved.

[0255] Domains with the ability to form oligomers or polymers can be used to increase the affinity of the variant antigen-binding domains of the present invention for TRBC2. Accordingly, the variant antigen-binding domains of the present invention can further comprise an oligomerisation domain. As used herein, the term "oligomerisation domain" refers to a domain that self-associates to form an oligomer (such as a dimer or trimer) or a polymer. Thus, as used herein, the term oligomerisation domain also refers to a polymerisation domain. Oligomerisation domains are well known to those skilled in the art, and any oligomerisation domain can be used in conjunction with the variant antigen-binding domains of the present invention, provided that the resulting oligomer maintains or improves the affinity of the monomeric variant antigen-binding domain for TRBC2. Examples of oligomerisation domains include, but are not limited to, the Fc region, the COMP spacer of SEQ ID NO: 18, or the truncated COMP as described in the context of the chimeric antigen receptor (CAR) of the present invention below.

[0256] The present invention also contemplates different forms of the variant antigen-binding domains of the present invention, including but not limited to scFv, diabody, trimerbody, minibody, F(ab) and F(ab') 2 fragments, as well as intact antibodies, namely IgG, IgM, IgA, IgD, IgE.

[0257] Accordingly, another aspect of the present invention relates to an antibody comprising the variant antigen-binding domain of the present invention, hereinafter referred to as " Antibody of the present invention ".

[0258] 3. Chimeric Antigen Receptor

[0259] In another aspect, the present invention provides a chimeric antigen receptor (CAR), hereinafter referred to as " The CAR of the present invention ", which comprises the variant antigen-binding domain of the present invention, a transmembrane domain and an intracellular domain.

[0260] The term "variant antigen-binding domain of the present invention" has been described in detail in the context of the first aspect of the present invention, and its features and embodiments are equally applicable to this aspect of the present invention.

[0261] As used herein, the term " Chimeric antigen receptor " or " CAR " or " Chimeric T cell receptor " or " Artificial T cell Receptor " or " Chimeric immune receptorrefers to a chimeric type I transmembrane protein that links an extracellular antigen recognition domain (the conjugate) to an intracellular signaling domain (the intracellular domain). The conjugate is usually a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms that contain an antigen-binding site. A spacer domain is usually necessary to separate the conjugate from the membrane and allow it to assume an appropriate orientation. A common spacer domain used is the Fc of IgG1. Depending on the antigen, a more compact spacer can suffice, such as the stalk from CD8α and even just the IgG1 hinge. The transmembrane domain anchors the protein in the cell membrane and links the spacer to the intracellular domain.

[0262] Early CAR designs had an intracellular domain derived from the intracellular portion of the γ chain of FcεR1 or CD3ζ. As a result, these first-generation receptors transmitted immune signal 1, which was sufficient to trigger T cell killing of associated target cells but not sufficient to fully activate T cells to proliferate and survive. To overcome this limitation, composite intracellular domains were constructed: the intracellular portion of a T cell co-stimulatory molecule was fused to the intracellular portion of CD3ζ, creating second-generation receptors that were able to transmit both activation and co-stimulatory signals upon antigen recognition. The co-stimulatory domain most commonly used is the co-stimulatory domain of CD28. This provides the most potent co-stimulatory signal, i.e., immune signal 2, which triggers T cell proliferation. A number of receptors have also been described that include intracellular domains of the TNF receptor family, such as the closely related OX40 and 4-1BB, which transmit survival signals. Even more potent third-generation CARs have now been described that have an intracellular domain capable of transmitting activation, proliferation, and survival signals.

[0263] When the CAR binds to the target antigen, this results in the transmission of an activation signal to the T cell on which it is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell to tumor cells expressing the targeted antigen.

[0264] Thus, a CAR generally comprises: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that contains a signaling domain or is associated with a signaling domain (see Figure 4 ).

[0265] A CAR can have the following general structure:

[0266] Antigen-binding domain - spacer domain - transmembrane domain - intracellular signaling domain (intracellular domain).

[0267] 3.1. Signal peptide

[0268] The CARs of the present invention can include a signal peptide such that when the CAR is expressed within a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface on which it is expressed.

[0269] The core of the signal peptide may contain a long stretch of hydrophobic amino acids, which tend to form a single α-helix. The signal peptide may start with a short stretch of positively charged amino acids, which helps to implement the correct topology of the polypeptide during translocation. At the end of the signal peptide, typically there is a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase can cleave during translocation or after completion to generate a free signal peptide and a mature protein. Then, the free signal peptide is digested by specific proteases.

[0270] The signal peptide can be at the amino terminus of the molecule.

[0271] The signal peptide may comprise SEQ ID NO: 3 to 5 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), provided that the signal peptide still has the function of causing cell surface expression of the protein.

[0272] SEQ ID NO:3: MGTSLLCWMALCLLGADHADG

[0273] The signal peptide of SEQ ID NO: 3 is compact and efficient. It is expected that about 95% cleavage occurs after the terminal glycine, thus being effectively removed by signal peptidase.

[0274] SEQ ID NO:4: MSLPVTALLLPLALLLHAARP

[0275] The signal peptide of SEQ ID NO: 4 is derived from IgG1.

[0276] SEQ ID NO:5: MAVPTQVLGLLLLWLTDARC

[0277] The signal peptide of SEQ ID NO: 5 is derived from CD8.

[0278] 3.2. Spacer domain

[0279] The CAR contains a spacer sequence to link the antigen-binding domain and the transmembrane domain and spatially separate the antigen-binding domain from the intracellular domain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate antigen binding.

[0280] In the CAR of the present invention, the spacer sequence can, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a human CD8 stalk or a murine CD8 stalk. Alternatively, the spacer can comprise an alternative linker sequence having length and / or domain spacing characteristics similar to those of an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk. The human IgG1 spacer can be modified to remove the Fc binding motif. The spacer can include a coiled-coil domain, such as that described in WO2016 / 151315.

[0281] The CAR of the present invention can comprise a sequence selected from the sequences shown in SEQ ID NOs: 6 to 10 or a variant thereof having at least 80% sequence identity.

[0282] SEQ ID NO:6 (hinge-CH2CH3 of human IgG1)

[0283] AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

[0284] SEQ ID NO:7 (human CD8 stalk):

[0285] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0286] SEQ ID NO:8 (human IgG1 hinge):

[0287] AEPKSPDKTHTCPPCPKDPK

[0288] SEQ ID NO:9 (CD2 extracellular domain)

[0289] KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITH

[0290] KWTTSLSAKFKCTAGNKVSKESSVEPVSCP

[0291] EKGLD

[0292] SEQ ID NO:10 (CD34 extracellular domain)

[0293] SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0294] SEQ ID NO:19 (COMP)

[0295] DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACG

[0296] The COMP coiled-coil domain can be truncated at the N-terminus while retaining surface expression. Thus, the coiled-coil COMP spacer can contain or consist of a truncated form of SEQ ID NO:18 truncated at the N-terminus. The truncated COMP can contain the 5 C-terminal amino acids of SEQ ID NO:19, i.e., the sequence CDACG (SEQ ID NO:20). The truncated COMP can contain 5 to 44 amino acids, such as at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. The truncated COMP can correspond to the C-terminus of SEQ ID NO:19. For example, a truncated COMP containing 20 amino acids can contain the sequence QQVREITFLKNTVMECDACG (SEQ ID NO:21). The truncated COMP can retain the cysteine residues involved in polymerization. The truncated COMP can retain the ability to form multimers.

[0297] 3.3. Transmembrane domain

[0298] The transmembrane domain is the sequence of the CAR that spans the membrane.

[0299] A transmembrane domain can be any protein structure that is thermodynamically stable in a membrane. This is typically an α-helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the present invention. The presence and span of the transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Additionally, considering that the transmembrane domain of a protein is a relatively simple structure, i.e., a polypeptide sequence predicted to form a hydrophobic α-helix long enough to span the membrane, an artificially designed TM domain (synthetic transmembrane components are described in US7052906 B1) can also be used.

[0300] The transmembrane domain can be derived from CD28, CD8a, or TYRP-1, which provide good receptor stability.

[0301] In one embodiment, the transmembrane domain is derived from CD8a.

[0302] SEQ ID NO:11: CD8a transmembrane domain

[0303] IYIWAPLAGTCGVLLLSLVIT

[0304] In another embodiment, the transmembrane domain is derived from TYRP-1.

[0305] SEQ ID NO:12: TYRP-1 transmembrane domain

[0306] IIAIAVVGALLLVALIFGTASYLI

[0307] 3.4. Intracellular domain

[0308] The intracellular domain is the signal transmission part of the CAR. After antigen recognition, receptor clusters, native CD45, and CD148 are expelled from the synapse and signal is transmitted to the cell. The most commonly used intracellular domain component is the intracellular domain component of CD3ζ containing 3 ITAMs. After antigen binding, it transmits an activation signal to T cells. CD3ζ may not provide a fully effective activation signal, so other co-stimulatory signalings may be required. Examples of co-stimulatory domains include the intracellular domains from CD28, OX40, 4-1BB, CD27, and ICOS, which can be used together with CD3ζ to transmit proliferation / survival signals.

[0309] In one embodiment, at least one co-stimulatory intracellular domain is used together with CD3ζ. In a specific embodiment, the co-stimulatory intracellular domain is selected from the intracellular domains of CD28, OX40, 4-1BB, CD27, and ICOS.

[0310] In another embodiment, at least two co-stimulatory intracellular domains are used together with CD3ζ. In certain embodiments, the two co-stimulatory intracellular domains are selected from the intracellular domains of CD28, OX40, 4-1BB, CD27, and ICOS in any combination and order. Particularly suitable combinations include the intracellular domains of CD28 and CD3ζ, the intracellular domains of OX40 and CD3ζ, the intracellular domains of 4-1BB and CD3ζ, the intracellular domains of CD28, OX40, and CD3ζ, and the intracellular domains of CD28, 4-1BB, and CD3ζ.

[0311] The transmembrane domain and the intracellular T cell signaling domain (intracellular domain) of a CAR having an activated intracellular domain can comprise a sequence as shown in SEQ ID NOs: 13 to 18 or a variant thereof having at least 80% sequence identity.

[0312] SEQ ID NO: 13 comprises the CD28 transmembrane domain and the CD3ζ intracellular domain

[0313] FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0314] SEQ ID NO: 14 comprises the CD28 transmembrane domain and the CD28 and CD3ζ intracellular domains

[0315] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0316] SEQ ID NO:15 contains the CD28 transmembrane domain and the intracellular domains of CD28, OX40, and CD3ζ: FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0317] SEQ ID NO:16 contains the CD8a transmembrane domain and the CD3ζ intracellular domain

[0318] IYIWAPLAGTCGVLLLSLVITRVLYCKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0319] SEQ ID NO:17 contains the CD8a transmembrane domain and the intracellular domains of 4-1BB and CD3ζ

[0320] IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0321] SEQ ID NO:18 contains the TYRP-1 transmembrane domain and the intracellular domains of 4-1BB and CD3ζ

[0322] IIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0323] The variant sequence can have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NOs: 13 to 18, provided that the sequence provides a valid transmembrane domain and a valid intracellular T cell signaling domain.

[0324] The CAR of the present invention can comprise a sequence selected from the sequences shown in SEQ ID NOs: 25 to 36.

[0325] SEQ ID NO:25: CAR comprising T28K, Y32F, A100N mutations in the VH domain, N35K mutation in the VL domain, IgG1 hinge spacer, TYRP-1 transmembrane domain, and 4-1BB and CD3ζ intracellular domains

[0326] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGKTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0327] SEQ ID NO:26: CAR comprising T28K, Y32F, A100N mutations in the VH domain, an IgG1 hinge spacer region, a TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0328] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0329] SEQ ID NO:27: CAR comprising T28K, Y32F, A100N, N103L mutations in the VH domain, an IgG1 hinge spacer region, a TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0330] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYLFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0331] SEQ ID NO:28: CAR containing the T28K, Y32F, A100N, N103M mutations in the VH domain, the N35Y mutation in the VL domain, an IgG1 hinge spacer, a TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0332] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGYTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0333] SEQ ID NO:29: CAR containing the T28K, Y32F, A100N, N103M mutations in the VH domain, the N35R mutation in the VL domain, an IgG1 hinge spacer, a TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0334] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGFMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0335] SEQ ID NO:30: CAR containing the T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain, the N35R mutation in the VL domain, an IgG1 hinge spacer, a TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0336] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGLMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0337] SEQ ID NO: 31: CAR containing the T28K, Y32F, A100N mutations in the VH domain, the CD8 stalk spacer, the TYRP-1 transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ

[0338] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0339] SEQ ID NO:32: CAR containing T28K, Y32F, A100N mutations in the VH domain, CD8 stalk spacer, TYRP-1 transmembrane domain, and CD28 and CD3ζ intracellular domains

[0340] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIIAIAVVGALLLVALIFGTASYLIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0341] SEQ ID NO:33: CAR containing T28K, Y32F, A100N mutations in the VH domain, IgG1 hinge spacer, TYRP-1 transmembrane domain, and CD28 and CD3ζ intracellular domains

[0342] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0343] SEQ ID NO: 34: CAR containing T28K, Y32F, A100N mutations in the VH domain, N35K mutation in the VL domain, IgG1 hinge spacer, TYRP-1 transmembrane domain, and CD28 and CD3ζ intracellular domains

[0344] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGKTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKIIAIAVVGALLLVALIFGTASYLIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0345] SEQ ID NO:35: CAR containing T28K, Y32F, A100N mutations in the VH domain, N35K mutation in the VL domain, CD8 stem spacer, TYRP-1 transmembrane domain, and 4-1BB and CD3ζ intracellular domains

[0346] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGKTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIIAIAVVGALLLVALIFGTASYLIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0347] SEQ ID NO:36: CAR containing the T28K, Y32F, A100N mutations in the VH domain, the N35K mutation in the VL domain, the CD8 stem spacer, the TYRP-1 transmembrane domain, and the CD28 and CD3ζ intracellular domains

[0348] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGYNFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGKTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIIAIAVVGALLLVALIFGTASYLIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0349] 4. Bispecific T Cell Engager

[0350] Based on the basic concept of having two antibody-like binding domains, a variety of molecules have been developed.

[0351] Bispecific T cell engagers are a class of bispecific antibody-like molecules that have been developed and are mainly used as anti-cancer drugs. They direct the cytotoxic activity of the host's immune system, more specifically of T cells, against target cells such as cancer cells. In these molecules, one binding domain binds to T cells via the CD3 receptor, while the other (via a tumor-specific molecule) binds to the target cell (such as a tumor cell). Since the bispecific molecule binds both the target cell and the T cell simultaneously, it brings the target cell into proximity with the T cell, enabling the T cell to exert its effects, such as cytotoxic effects on cancer cells. The formation of the T cell:bispecific Ab:cancer cell complex induces signal transduction in the T cell, leading to the release of, for example, cytotoxic mediators. Ideally, the reagent induces the desired signal transduction only in the presence of the target cell, resulting in selective killing.

[0352] A variety of different forms of bispecific T cell engagers have been developed, but the most common one is a fusion consisting of two tandemly arranged single-chain variable fragments (scFv) of different antibodies. These are sometimes called BiTE (bispecific T cell engager).

[0353] The present invention also contemplates bispecific molecules that selectively recognize TRBC2 and are capable of activating T cells. For example, the molecule can be a BiTE.

[0354] Thus, in another aspect, the present invention provides a bispecific T cell engager (BiTE), hereinafter referred to as " The present invention BiTE of ", which comprises a variant antigen-binding domain of the present invention and a T cell activation domain.

[0355] The term "variant antigen-binding domain of the present invention" has been described in detail in the context of the first aspect of the present invention, and its features and embodiments are equally applicable to this aspect of the present invention.

[0356] As used herein, the term " T cell activation domain " refers to a second domain capable of activating T cells. The T cell activation domain can be a scFv that specifically binds to CD3. Examples of anti-CD3 scFvs suitable for the purposes of the present invention are well known in the art and include, but are not limited to, scFvs derived from OKT3.

[0357] The bispecific molecule can comprise a signal peptide to assist in its production. The signal peptide can cause the bispecific molecule to be secreted by the host cell, such that the bispecific molecule can be harvested from the host cell supernatant.

[0358] The signal peptide can be at the amino terminus of the molecule. The bispecific molecule can have the general formula: signal peptide - variant antigen-binding domain of the present invention - T cell activation domain.

[0359] The bispecific molecule can comprise a spacer sequence to link the variant antigen-binding domain of the present invention to the T cell activation domain and spatially separate the two domains.

[0360] The spacer sequence can, for example, comprise an IgG1 hinge or a CD8 stalk. The linker can alternatively comprise an alternative linker sequence having a length and / or domain-spacing property similar to that of the IgG1 hinge or CD8 stalk.

[0361] 5. Nucleic Acid

[0362] In another aspect, the present invention also provides a nucleic acid sequence encoding the variant antigen-binding domain of the present invention, hereinafter referred to as " The first nucleic acid of the present invention ".

[0363] In another aspect, the present invention also provides a nucleic acid sequence encoding the antibody of the present invention, hereinafter referred to as " The second nucleic acid of the present invention ".

[0364] In another aspect, the present invention also provides a nucleic acid sequence encoding the CAR of the present invention, hereinafter referred to as " The third nucleic acid of the present invention ".

[0365] In another aspect, the present invention also provides a nucleic acid sequence encoding the BiTE of the present invention, hereinafter referred to as " The fourth nucleic acid of the present invention ".

[0366] The terms "variant antigen-binding domain of the present invention", "antibody of the present invention" and "BiTE of the present invention" have been described in detail in the context of the previous aspects of the present invention, and their features and embodiments are equally applicable to these aspects of the present invention.

[0367] As used herein, the terms " Polynucleotide ", " Nucleotide " and " Nucleic acid " are intended to be synonymous with each other.

[0368] Those skilled in the art will understand that due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Additionally, it should be understood that those skilled in the art can use conventional techniques for nucleotide substitution, which do not affect the polypeptide sequence encoded by the polynucleotides described herein, to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0369] To avoid homologous recombination, the nucleic acid sequences and constructs of the present invention may contain alternative codons in sequence regions encoding the same or similar amino acid sequences.

[0370] The nucleic acids according to the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that include synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0371] The terms " Variant ", " Homolog " or " Derivative " in relation to a nucleotide sequence include any substitution, variation, modification, replacement, deletion or addition of one (or more) nucleic acids of the sequence.

[0372] 6. Vector

[0373] The present invention also provides a vector, or a kit of vectors, which comprises one or more nucleic acid sequences of the present invention. Such vectors can be used to introduce nucleic acid sequences into host cells such that they express a variant antigen-binding molecule or antibody or CAR or BiTE of the present invention.

[0374] The terms "variant antigen-binding domain of the present invention", "antibody of the present invention" and "BiTE of the present invention" have been described in detail in the context of the previous aspects of the present invention, and their features and embodiments are equally applicable to these aspects of the present invention.

[0375] The vector can be, for example, a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0376] The vector can be capable of transfecting or transducing lytic immune cells, such as T cells or NK cells.

[0377] 7. Cell

[0378] Another aspect of the present invention relates to a cell, hereinafter referred to as " Cell of the present invention ", which comprises a CAR of the present invention.

[0379] The cell can comprise a nucleic acid or a vector of the present invention.

[0380] The terms "CAR of the present invention", "nucleic acid of the present invention", "vector of the present invention" have been described in detail in the context of the previous aspects of the present invention, and their features and embodiments are equally applicable to these aspects of the present invention.

[0381] The cell can be a lytic immune cell, such as a T cell or an NK cell.

[0382] T cells, or T lymphocytes, are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their cell surface. There are multiple types of T cells, as summarized below.

[0383] Helper T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. They are activated when peptide antigens are presented to them by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9 or TFH, which secrete different cytokines to promote different types of immune responses.

[0384] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I present on the surface of all nucleated cells. CD8+ cells can be inactivated to an incompetent state by IL-10, adenosine, and other molecules secreted by regulatory T cells, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0385] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has been resolved. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells contain three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0386] Regulatory T cells (Treg cells), formerly called suppressor T cells, are crucial for maintaining immune tolerance. Their main role is to turn off T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus.

[0387] Two major types of CD4+ Treg cells have been described, namely naturally occurring Treg cells and adaptive Treg cells.

[0388] Naturally occurring Treg cells (also called CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been associated with interactions between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, leading to the fatal autoimmune disease IPEX.

[0389] Adaptive Treg cells (also called Tr1 cells or Th3 cells) can arise during normal immune responses.

[0390] The cells can be natural killer cells (or NK cells). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0391] NK cells (which belong to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third cell type that differentiates from a common lymphoid progenitor cell that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they enter the circulation.

[0392] The cells of the present invention can be any of the above cell types. In one embodiment, the cells of the present invention are T cells. In another embodiment, the cells of the present invention are NK cells.

[0393] The cells according to this aspect of the present invention can be created ex vivo from the patient's own peripheral blood (first party), or in the context of a hematopoietic stem cell graft (second party) from a donor's peripheral blood, or from a peripheral blood of an unrelated donor (third party).

[0394] Alternatively, the cells according to this aspect of the present invention can be derived from inducible progenitor cells or embryonic progenitor cells that differentiate ex vivo into lytic cells. Alternatively, an immortalized lytic cell line that retains its lytic function and can act as a therapeutic agent, such as a T or NK cell, can be used.

[0395] In all these embodiments, CAR-expressing cells are generated by introducing DNA or RNA encoding a chimeric polypeptide by one of many means including transduction with a viral vector, transfection with DNA or RNA.

[0396] The cells of the present invention can be isolated cells from a subject. The cells can be from a peripheral blood mononuclear cell (PBMC) sample. The cells, particularly lytic cells such as T or NK cells, can be activated and / or expanded before transduction with a nucleic acid encoding a molecule providing the CAR of the present invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0397] The cells of the present invention can be prepared by a method that includes the step of transducing or transfecting the cells with a vector of the present invention that contains a nucleic acid sequence encoding a CAR.

[0398] Before the transduction or transfection step, the method for preparing the cells of the present invention can further include the step of isolating the cells from a cell-containing sample from a subject or other sources listed above. In the case where the cells are lytic cells, the sample is a cell-containing sample from a subject that contains lytic cells.

[0399] In the present invention On used hereinafter The the term Subject ” or “ Individual ” refers to a member of a mammalian species, preferably male or female of any age or race.

[0400] The cells of the invention can then be purified, for example, by selection based on the expression of a CAR-based antigen-binding domain.

[0401] 8. Conjugate

[0402] The variant antigen-binding domains or antibodies of the invention can be conjugates of variant antigen-binding domains or antibodies, for example, the conjugate can be a detectable entity or a chemotherapeutic entity.

[0403] The terms "variant antigen-binding domain of the invention" and "antibody of the invention" have been described in detail in the context of the previous aspects of the invention, and their features and embodiments are equally applicable to these aspects of the invention.

[0404] The detectable entity can be a fluorescent moiety, such as a fluorescent peptide. As used herein, the term " Fluorescent peptide " refers to a polypeptide that emits light at a detectable wavelength upon excitation. Examples of fluorescent proteins include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), enhanced GFP, red fluorescent protein (RFP), blue fluorescent protein (BFP), and mCherry.

[0405] The variant antigen-binding domain or antibody of the invention conjugated to a detectable entity can be used to determine the TRBC of malignant T cells.

[0406] As used herein, the term " Chemotherapeutic entity " refers to an entity that is destructive to cells, i.e., the entity reduces the viability of the cells. The resulting conjugate will hereinafter be referred to as " Chemotherapeutic conjugate of the present invention". The chemotherapeutic entity can be a cytotoxic drug. Chemotherapeutic entities contemplated include, but are not limited to, alkylating agents, nitrosoureas, ethyleneimines / methylmelamines, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophylotoxins, enzymes such as L-asparaginase; biologic response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; platinum coordination complexes such as cisplatin and carboplatin, anthracenediones, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocortical steroid antagonists such as prednisone and its equivalents, dexamethasone and aminoglutethimide; progesterones such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide.

[0407] The variant antigen-binding domain or antibody of the invention conjugated to a chemotherapeutic entity enables the targeted delivery of the chemotherapeutic entity to cells expressing TRBC2.

[0408] 9. Pharmaceutical Composition

[0409] The invention also relates to a pharmaceutical composition comprising the cells or plurality of cells, or antibody, or BiTE, or chemotherapeutic conjugate of the invention, hereinafter referred to as " Pharmaceutical composition of the present invention ".

[0410] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

[0411] The terms "cells of the invention", "antibodies of the invention", "BiTEs of the invention", and "chemotherapeutic conjugates of the invention" have been described in detail in the context of the previous aspects of the invention, and their characteristics and embodiments are equally applicable to these aspects of the invention.

[0412] Administration

[0413] Administration of one or more cells, or antibody, or BiTE, or chemotherapeutic conjugate of the invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable. For example, the agent can be administered orally and parenterally, intraperitoneally, intravenously, subcutaneously, transdermally, intramuscularly, via local delivery such as through a catheter or stent.

[0414] Typically, a physician will determine the actual dose that is most suitable for an individual subject, and that dose will vary with the age, weight, and response of the particular patient. The dose is sufficient to reduce or deplete the number of clonal T cells expressing TRBC1 or TRBC2.

[0415] 10. Treatment Method

[0416] In another aspect, the present invention provides the cells, or antibodies, or BiTEs, or chemotherapeutic conjugates of the present invention for use in a medicament.

[0417] In another aspect, the present invention provides a method for treating T cell lymphoma or leukemia in a subject, hereinafter referred to as " Treatment method of the present invention ", which comprises the step of administering to the subject the cells, or antibodies, or BiTEs, or chemotherapeutic conjugates of the present invention, wherein the malignant T cells express TRBC2. The administering step may be in the form of a pharmaceutical composition as described above.

[0418] This aspect of the present invention may alternatively be formulated as the cells, or antibodies, or BiTEs, or chemotherapeutic conjugates of the present invention for treating T cell lymphoma or leukemia, hereinafter referred to as " For the cells, antibodies, BiTEs, or chemotherapeutic conjugates of the present invention Chemotherapeutic conjugate ", wherein the malignant T cells express TRBC2.

[0419] This aspect of the present invention may alternatively be formulated as the use of the cells, or antibodies, or BiTEs, or chemotherapeutic conjugates of the present invention in the preparation of a medicament for treating T cell lymphoma or leukemia, wherein the malignant T cells express TRBC2.

[0420] The terms "cells of the present invention", "antibodies of the present invention", "BiTEs of the present invention", "subject", and "chemotherapeutic conjugates of the present invention" have been described in detail in the context of the previous aspects of the present invention, and their characteristics and embodiments are equally applicable to these aspects of the present invention.

[0421] The method for treating T cell lymphoma and / or leukemia involves the therapeutic use of the cells, antibodies, BiTEs, or chemotherapeutic agent conjugates of the present invention. Herein, the cells, antibodies, BiTEs, or chemotherapeutic agent conjugates of the present invention may be administered to a subject having an existing disease of T cell lymphoma and / or leukemia to alleviate, reduce, or improve at least one symptom associated with the disease and / or slow down, reduce, or arrest the development of the disease.

[0422] Methods for preventing T cell lymphoma and / or leukemia involve the prophylactic use of the cells, antibodies, BiTEs, or chemotherapeutic conjugates of the present invention. Herein, such cells, antibodies, BiTEs, or chemotherapeutic conjugates can be administered to a subject who has not yet developed T cell lymphoma and / or leukemia and / or does not exhibit any symptoms of T cell lymphoma and / or leukemia to prevent or attenuate the cause of the disease, or to reduce or prevent the development of at least one symptom associated with the disease. The subject may be susceptible to T cell lymphoma and / or leukemia or be considered at risk of developing T cell lymphoma and / or leukemia.

[0423] The methods may involve the steps of:

[0424] (i) isolating a sample containing cytotoxic cells;

[0425] (ii) transducing or transfecting the cells with a nucleic acid sequence or vector provided by the present invention; and

[0426] (iii) administering the cells from (ii) to a subject.

[0427] The sample containing cytotoxic cells can be isolated from a subject or from other sources such as those described above. Cytotoxic cells such as T cells or NK cells can be isolated from the patient's own peripheral blood (first party), or in the context of a hematopoietic stem cell graft from a donor's peripheral blood (second party), or from the peripheral blood of an unrelated donor (third party).

[0428] Methods for treating T cell lymphoma and / or leukemia involve the therapeutic use of a reagent. Herein, a reagent can be administered to a subject with an existing disease of T cell lymphoma and / or leukemia to alleviate, reduce, or improve at least one symptom associated with the disease and / or slow down, reduce, or prevent the progression of the disease.

[0429] These therapeutic applications will include administering a therapeutically effective amount of the cells, antibodies, BiTEs, or chemotherapeutic conjugates of the present invention.

[0430] As used herein, the term " Therapeutically effective amount " refers to the amount of the cells, antibodies, BiTEs, or chemotherapeutic conjugates of the present invention required to achieve a significant prevention, cure, delay, reduction in the severity of, or improvement in one or more symptoms of TRBC2-positive T cell lymphoma and / or leukemia.

[0431] The method of the present invention can be used to treat any lymphoma and / or leukemia associated with clonal expansion of cells expressing a T cell receptor (TCR) comprising TRBC2. Thus, the present invention relates to a method for treating diseases involving malignant T cells that express a TCR comprising TRBC2.

[0432] The method of the present invention can be used to treat T cell lymphomas in which malignant T cells express a TCR comprising TRBC2. “ Lymphoma ” is used herein according to its standard meaning to refer to a cancer that typically develops in lymph nodes but can also affect the spleen, bone marrow, blood, and other organs. Lymphomas typically present as solid tumors of lymphoid cells. The main symptom associated with lymphomas is lymphadenopathy, although secondary (B) symptoms can include fever, night sweats, weight loss, loss of appetite, fatigue, respiratory distress, and itching.

[0433] The method of the present invention can be used to treat T cell leukemias in which malignant T cells express a TCR comprising TRBC2. “ Leukemia ” is used herein according to its standard meaning to refer to a cancer of the blood or bone marrow.

[0434] The following is an illustrative, non-exhaustive list of diseases that can be treated by the method of the present invention.

[0435] Peripheral T cell lymphoma

[0436] Peripheral T cell lymphomas are relatively rare lymphomas and account for less than 10% of all non-Hodgkin lymphomas (NHL). However, they are associated with an aggressive clinical course, and the etiology and exact cell origin of most T cell lymphomas remain poorly defined.

[0437] Lymphomas typically first present as swelling in the neck, axilla, or groin. Additional swelling can occur in other locations where lymph nodes are located, such as the spleen. Typically, lymph node enlargement can invade blood vessels, nerves, or the space of the stomach, resulting in swollen arms and legs, tingling and numbness, or a feeling of fullness, respectively. Lymphoma symptoms also include non-specific symptoms such as fever, chills, unexplained weight loss, night sweats, lethargy, and itching.

[0438] The WHO classification uses morphological and immunophenotypic features combined with clinical aspects and, in some cases, genetics to describe prognostically and therapeutically relevant categories of peripheral T-cell lymphomas (Swerdlow et al.; WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed.; Lyon: IARC Press; 2008). The anatomic localization of neoplastic T cells is partly parallel to their proposed normal cell counterparts and functions, and thus T-cell lymphomas are associated with lymph nodes and peripheral blood. This approach allows for a better understanding of some manifestations of T-cell lymphomas, including their cellular distribution, some aspects of morphology, and even associated clinical findings.

[0439] The most common in T-cell lymphomas is peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), which accounts for 25% of the total, followed by angioimmunoblastic T-cell lymphoma (AITL) (18.5%).

[0440] Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS)

[0441] PTCL-NOS encompasses more than 25% of all peripheral T-cell lymphomas and NK / T-cell lymphomas and is the most common subtype. It is defined by exclusion and does not correspond to any specific mature T-cell lymphoma entity listed in the current WHO 2008. Thus, it is analogous to diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS).

[0442] Most patients are adults, with a median age of 60 and a male-to-female ratio of 2:1. Most cases originate in lymph nodes; however, extranodal presentation occurs in approximately 13% of patients and most commonly involves the skin and gastrointestinal tract.

[0443] The cytological spectrum is very broad, ranging from polymorphic to monomorphic. Three morphologically defined variants have been described, including the lymphoepithelioid (Lennert) variant, the T-zone variant, and the follicular variant. The lymphoepithelioid variant of PTCL contains abundant background epitheloid histiocytes and is usually positive for CD8. It is associated with a better prognosis. The follicular variant of PTCL-NOS is emerging as a potentially distinct clinicopathologic entity.

[0444] Most PTCL-NOS have a mature T-cell phenotype and are most often CD4-positive. Variable loss of at least one pan-T-cell marker (CD3, CD2, CD5, or CD7) is seen in 75% of cases, with CD7 and CD5 most often downregulated. CD30 can be expressed and CD15 is rarely expressed, where CD15 is an adverse prognostic feature. CD56 expression, while uncommon, also has a negative prognostic impact. Additional adverse pathologic prognostic factors include a proliferation rate greater than 25% based on KI-67 expression and the presence of more than 70% transformed cells. Immunophenotypic analysis of these lymphomas provides little insight into their biology. Angioimmunoblastic T-cell lymphoma (AITL)

[0445] AITL is a systemic disease characterized by a polymorphic infiltrate involving lymph nodes, prominent high endothelial venules (HEVs), and a meshwork of follicular dendritic cells (FDCs) with perivascular expansion. AITL is considered a re-T-cell lymphoma derived from follicular helper type (TFH) αβ T cells, which are typically found in germinal centers.

[0446] AITL is the second most common entity among peripheral T-cell lymphomas and NK / T-cell lymphomas, accounting for approximately 18.5% of cases. It occurs between middle age and the elderly, with a median age of 65 years and approximately equal incidence in males and females. Clinically, patients typically have advanced disease with generalized lymphadenopathy, hepatosplenomegaly, and prominent constitutional symptoms. A rash with pruritus is often present. Polyclonal hypergammaglobulinemia is frequent and is associated with autoimmune phenomena.

[0447] Three distinct morphologic patterns have been described in AITL. Early lesions of AITL (pattern I) typically show preserved architecture with characteristic hyperplastic follicles. Neoplastic proliferation is confined to the periphery of the follicles. In pattern II, the architecture of the node is partially obliterated, with a few regressed follicles preserved. The subcapsular sinus is retained and even dilated. The paracortex contains dendritic HEVs and there is proliferation of FDCs beyond B-cell follicles. The neoplastic cells are small to medium-sized, with minimal cytologic atypia. They typically have clear to pale eosinophilic cytoplasm and can show prominent T-cell membranes. A polymorphic inflammatory background is usually prominent.

[0448] Although AITL is a T-cell malignancy, there is a characteristic expansion of B cells and plasma cells, which may reflect the function of neoplastic cells as TFH cells. Both EBV-positive and EBV-negative B cells are present. Occasionally, atypical B cells can resemble Hodgkin / Reed-Sternberg-like cells morphologically and immunophenotypically, sometimes leading to diagnostic confusion for this entity. B-cell proliferation in AITL can be extensive, and some patients develop secondary EBV-positive diffuse large B-cell lymphoma (DLBCL), or less commonly EBV-negative B-cell tumors, usually with plasmacytic differentiation.

[0449] The neoplastic CD4-positive T cells in AITL show strong expression of CD10 and CD279 (PD-1), and are positive for CXCL13. CXCL13 leads to increased B-cell recruitment to lymph nodes via adhesion to HEV, B-cell activation, plasmacytic differentiation, and expansion of the FDC mesh, all of which contribute to the morphology and clinical features of AITL. Strong PD-1 expression in tumor cells in the perifollicular area particularly helps to distinguish AITL pattern I from reactive follicular and paracortical hyperplasia.

[0450] The follicular variant of PTCL-NOS is another entity with a TFH phenotype. In contrast to AITL, it does not have significant extrafollicular expansion of HEV or FDC mesh. Neoplastic cells can form intrafollicular aggregates mimicking B-cell follicular lymphoma, but can also have an interfollicular growth pattern or involve an expanded mantle zone. Clinically, the follicular variant of PTCL-NOS differs from AITL in that patients more often present with early disease, with partial lymph node involvement, and may lack the constitutional symptoms associated with AITL.

[0451] Anaplastic large cell lymphoma (ALCL)

[0452] ALCL can be subclassified into ALCL-“anaplastic lymphoma kinase” (ALK)+ or ALCL-ALK-.

[0453] ALCL-ALK+ is one of the best-defined entities within peripheral T-cell lymphomas, with characteristic “markers cells” with horseshoe-shaped nuclei that express ALK and CD30. It accounts for approximately 7% of all peripheral T-cell and NK-cell lymphomas and is most common in the first three decades of life. Patients often present with lymphadenopathy, but involvement of extranodal sites (skin, bone, soft tissue, lung, liver) and B symptoms are common.

[0454] ALCL, ALK+ shows a wide morphological spectrum in which five different patterns have been described, but all variants contain some hallmark cells. The hallmark cells have an eccentric horseshoe- or kidney-shaped nucleus and a prominent perinuclear eosinophilic Golgi region. Tumor cells grow in a cohesive pattern with a preference for sinus involvement. Smaller tumor cells predominate in the small cell variant, while in the lymphohistiocytic variant, abundant histiocytes obscure the presence of tumor cells, many of which are small.

[0455] By definition, all cases show ALK and CD30 positivity, with expression usually weaker in the smaller tumor cells. Pan-T cell markers are often lost, with surface expression of CD3 lacking in 75% of cases.

[0456] ALK expression is the result of a characteristic recurrent genetic alteration consisting of rearrangement of the ALK gene on chromosome 2p23 to one of many partner genes, leading to expression of a chimeric protein. The most common partner gene, present in 75% of cases, is nucleophosmin (NPM1) on chromosome 5q35, resulting in t(2;5)(p23;q35). The cellular distribution of ALK in different translocation variants can depend on the partner gene.

[0457] ALCL-ALK- is included in the 2008 WHO classification as a provisional category. It is defined as a CD30-positive T-cell lymphoma that is morphologically indistinguishable from ALCL-ALK+ by its cohesive growth pattern and the presence of hallmark cells, but lacks ALK protein expression.

[0458] Patients are usually adults between the ages of 40 and 65, in contrast to ALCL-ALK+ which is more common in children and young adults. ALCL-ALK- can involve both lymph nodes and extranodal tissues, although the latter is less common than in ALCL-ALK+. Most cases of ALCL-ALK- show effacement of the nodal architecture by sheets of cohesive neoplastic cells with typical "hallmark" features. In contrast to ALCL-ALK+, small cell morphological variants are not recognized.

[0459] Unlike its ALK+ counterpart, ALCL-ALK- shows more preservation of surface T cell marker expression, while expression of cytotoxic markers and epithelial membrane antigen (EMA) is less likely. Gene expression signatures and recurrent chromosomal imbalances are different in ALCL-ALK- and ALCL-ALK+, confirming that they are distinct entities at the molecular and genetic levels.

[0460] ALCL-ALK- is clinically distinct from both ALCL-ALK+ and PTCL-NOS, and there are significant differences in prognosis among these three distinct entities. The 5-year overall survival rate of ALCL-ALK- has been reported to be 49%, which is inferior to that of ALCL-ALK+ (70%), but is significantly better than that of PTCL-NOS (32%).

[0461] Enteropathy-associated T-cell lymphoma (EATL)

[0462] EATL is an aggressive neoplasm that is thought to be derived from intraepithelial T cells of the intestine. Two morphologically, immunohistochemically, and genetically distinct types of EATL were recognized in the 2008 WHO classification: type I (representing the majority of EATL) and type II (comprising 10–20% of cases).

[0463] Type I EATL is usually associated with overt or clinically silent gluten-sensitive enteropathy and is more common in Nordic patients because of the high prevalence of celiac disease in this population.

[0464] The lesions of EATL are most commonly found in the jejunum or ileum (90% of cases) and rarely in the duodenum, colon, stomach, or extra-gastrointestinal sites. The intestinal lesions are usually multifocal with mucosal ulceration. The clinical course of EATL is aggressive, and most patients die of the disease or its complications within 1 year.

[0465] The cytological spectrum of type I EATL is broad and can contain anaplastic cells in some cases. There is a polymorphic inflammatory background that can mask the neoplastic component in some cases. The intestinal mucosa in the area adjacent to the tumor often shows features of celiac disease, with blunted villi and an increased number of intraepithelial lymphocytes (IELs), which may represent the precursor cells of the lesion.

[0466] By immunohistochemistry, the neoplastic cells are usually CD3+CD4-CD8-CD7+CD5-CD56-βF1+ and contain cytotoxic granule-associated proteins (TIA-1, granzyme B, perforin). CD30 is partially expressed in almost all cases. CD103 is a mucosal homing receptor and can be expressed in EATL.

[0467] Type II EATL, also known as monomorphic CD56+ intestinal T-cell lymphoma, is defined as an intestinal neoplasm composed of small to medium-sized monomorphic T cells that express both CD8 and CD56. There is often a transverse spread of the tumor within the mucosa, and there is no inflammatory background. Most cases express γδTCR, but cases associated with αβTCR also exist.

[0468] Type II EATL has a more widespread distribution than type I EATL and often occurs in Asian or Hispanic groups in whom celiac disease is rare. Among individuals with EATL of European ancestry, type II represents approximately 20% of intestinal T-cell lymphomas, and a history of celiac disease is present in at least a subset of cases. The clinical course is aggressive.

[0469] Hepatosplenic T-cell lymphoma (HSTL)

[0470] HSTL is an aggressive systemic neoplasm that typically derives from the γδ cytotoxic T cells of the innate immune system, although in rare cases it may also derive from αβ T cells. It is one of the rarest T-cell lymphomas and typically affects adolescents and young adults (median age 35 years), with a male predominance.

[0471] Extranodal NK / T-cell lymphoma, nasal type

[0472] Extranodal NK / T-cell lymphoma, nasal type is an aggressive disease that typically has destructive midline lesions and necrosis. Most cases are NK-cell derived, but some cases derive from cytotoxic T cells. It is commonly associated with Epstein–Barr virus (EBV).

[0473] Cutaneous T-cell lymphoma

[0474] The method of the present invention can also be used to treat cutaneous T-cell lymphoma.

[0475] Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells to the skin, which results in the appearance of various lesions. These lesions change shape as the disease progresses, typically starting as a rash and eventually forming plaques and tumors, which then spread to other parts of the body.

[0476] Cutaneous T-cell lymphoma includes those mentioned in the following illustrative, non-exhaustive list; mycosis fungoide, pagetoid reticulosis, Sézary syndrome, granulomatous slack skin, lymphomatoid papulosis, pityriasis lichenoides chronica, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30− cutaneous large T-cell lymphoma, polymorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, and angiocentric lymphoma.

[0477] The signs and symptoms of CTCL vary according to the specific disease, and the two most common types are mycosis fungoide and Sézary syndrome. Classic mycosis fungoide is divided into three stages:

[0478] - Patch (atrophic or non-atrophic): patches on the non-specific dermatitis, lower trunk and buttocks; minimal / no pruritus;

[0479] - Plaque: severely pruritic plaques, lymphadenopathy; and

[0480] - Tumor: prone to ulceration

[0481] Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include skin edema, lymphadenopathy, hyperkeratosis of the palms and / or soles, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly.

[0482] Among all primary cutaneous lymphomas, 65% are of the T-cell type. The most common immunophenotype is CD4 positive. These diseases do not have a common pathophysiology as the term cutaneous T-cell lymphoma encompasses a variety of conditions.

[0483] The main etiological mechanism underlying the development of cutaneous T-cell lymphoma (i.e., mycosis fungoides) has not been elucidated. Mycosis fungoides may precede T-cell-mediated chronic inflammatory skin diseases, which can occasionally progress to a fatal lymphoma.

[0484] Primary cutaneous ALCL (C-ALCL)

[0485] Morphologically, C-ALCL is usually indistinguishable from ALC-ALK-. It is defined as a cutaneous tumor of large cells with anaplastic, pleomorphic, or immunoblastic morphology, in which more than 75% of the cells express CD30. C-ALCL, together with lymphomatoid papulosis (LyP), belongs to the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders, which as a group includes the second most common group of cutaneous T-cell lymphoproliferative disorders after mycosis fungoides.

[0486] The immunohistochemical staining profile is very similar to ALCL-ALK-, in which cytotoxic markers stain positively in the majority of cases. At least 75% of the tumor cells should be positive for CD30. CD15 may also be expressed and, when lymph node involvement occurs, may be indistinguishable from classical Hodgkin lymphoma. Rare cases of ALCL-ALK+ may present as local skin lesions and may resemble C-ALCL. T-cell acute lymphoblastic leukemia

[0487] T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% and 25% of ALL in the pediatric and adult populations, respectively. Patients usually have a high white blood cell count and may present with organomegaly, especially mediastinal widening and CNS involvement.

[0488] The method of the present invention can be used to treat T-ALL associated with malignant T cells expressing a TCR comprising TRBC.

[0489] T-cell prolymphocytic leukemia

[0490] T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell leukemia with aggressive behavior and a predilection for blood, bone marrow, lymph nodes, liver, spleen, and skin involvement. T-PLL mainly affects adults over 30 years of age. Other names include T-cell chronic lymphocytic leukemia, "knobby" type T-cell leukemia, and T-prolymphocytic leukemia / T-cell lymphocytic leukemia.

[0491] In peripheral blood, T-PLL consists of medium-sized lymphocytes with a single nucleolus and basophilic cytoplasm with occasional blebs or projections. The nucleus is usually round to oval, with occasional patients having cells with a more irregular nuclear contour, similar to the cerebriform nuclear shape seen in Sézary syndrome. The small cell variant comprises 20% of all T-PLL cases, while the Sézary cell (cerebriform) variant is seen in 5% of cases.

[0492] T-PLL has the immunophenotype of mature (post-thymic) T lymphocytes, and the neoplastic cells are usually positive for the pan-T antigens CD2, CD3, and CD7, and negative for TdT and CD1a. The immunophenotype CD4+ / CD8- is present in 60% of cases, the CD4+ / CD8+ immunophenotype in 25% of cases, and the CD4- / CD8+ immunophenotype in 15% of cases.

[0493] The T-cell lymphoma or leukemia to be treated or prevented can be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma, nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0494] The treatment method can include the step of administering a therapeutically effective amount of the cells or plurality of cells, or antibody, or BiTE, or chemotherapeutic conjugate of the present invention. A person skilled in the art will be able to determine, by conventional methods, the amount of the cells or plurality of cells, or antibody, or BiTE, or chemotherapeutic conjugate of the present invention that can exert a therapeutic effect on a patient.

[0495] 11. Diagnostic Agent

[0496] It has been previously determined that TRBC1 from healthy donors+ and TRBC2 + The proportions of T cells were 35% and 65% respectively, i.e., the median percentage of the total number of T cells expressing TRBC1 was 35% (range 25 - 47%) (Maciocia et al., 2017, Nat Med 23:1416 - 23). Since T cell lymphoma or leukemia is a clonal cancer (Maciocia et al., 2017; as above), the dysregulated malignant T cell proliferation characteristic of T cell lymphoma or leukemia will result in + either TRBC1 + or TRBC2 - T cells (i.e., TRBC2 - or TRBC1

[0497] Therefore, in another aspect, the present invention provides a diagnostic agent, hereinafter referred to as " The first diagnostic agent of the present invention ", which comprises a variant antigen-binding domain of the present invention.

[0498] In another aspect, the present invention provides a diagnostic agent, hereinafter referred to as " The second diagnostic agent of the present invention ", which comprises an antibody variant of the present invention. The terms "variant antigen-binding domain of the present invention" and "antibody of the present invention" have been described in detail in the context of the previous aspects of the present invention, and their characteristics and embodiments are equally applicable to these aspects of the present invention.

[0499] The variant antigen-binding domain or antibody of the present invention used in these assays can be labeled or unlabeled. The term " Detectable label " or " Labeling agent" refers to a molecular marker that allows the detection, localization, and / or discrimination of the molecule to which it is attached using suitable procedures and devices for detection (e.g., by spectroscopic, photochemical, biochemical, immunochemical, or chemical methods). Labeling agents suitable for labeling antibodies include radionuclides, enzymes, fluorophores, chemiluminescent agents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and derivatives, etc. As will be understood by those skilled in the art, unlabeled variant antigen-binding domains and antibodies need to be detected using additional reagents, e.g., labeled secondary antibodies. This is particularly useful for increasing the sensitivity of detection methods because it allows the signal to be amplified. There are a variety of conventional assays that can be used in the present invention, which use unlabeled antibodies of the present invention (primary antibodies) and labeled antibodies of the present invention (secondary antibodies); these techniques include Western blotting or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry, and immunohistochemistry techniques, flow cytometry or multiplex detection techniques based on the use of protein microspheres, biochips, or microarrays including antibodies of the present invention. Other methods for detecting and quantifying TRBC2 using the variant antigen-binding domains or antibodies of the present invention include affinity chromatography techniques or ligand-binding assays.

[0500] The diagnostic agent can be used for diagnosing T cell lymphoma or leukemia.

[0501] T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0502] 12. Diagnostic Method

[0503] In another aspect, the present invention provides a method for diagnosing T cell lymphoma or leukemia in a subject, hereinafter referred to as " Diagnostic method of the present invention ", which comprises the step of contacting a variant antigen-binding domain or antibody of the present invention with a sample containing T cells from the subject.

[0504] The terms "variant antigen-binding domain of the present invention", "antibody of the present invention", and "subject" have been described in detail in the context of the previous aspects of the present invention, and their characteristics and embodiments are equally applicable to this aspect of the present invention.

[0505] The diagnostic method of the present invention may further include the step of determining the percentage of TRBC2-positive T cells in the sample.

[0506] To practice the first method of the present invention, a sample containing T cells, such as a biological sample, is obtained from a subject to be studied. As used herein, the terms "sample" or "biological sample" include different types of biological fluids or tissue sections of affected organs containing T cells. Illustrative non-limiting examples of samples that can be used in the diagnostic method of the present invention include different types of biological fluids containing T cells, such as blood, lymph fluid, and cerebrospinal fluid. These biological fluid samples can be obtained by any conventional method known to those skilled in the art. Alternatively, the sample can also be, for example, a section of an affected organ tissue sample from a lymph gland, spleen, tonsil, or thymus, which can be obtained by any conventional method, such as by biopsy or surgical resection, as well as cryosections for histological purposes.

[0507] In the first step of the diagnostic method according to the present invention, the variant antigen-binding domain or antibody of the present invention is contacted with a sample from the subject under study under suitable conditions known to those skilled in the art.

[0508] Those skilled in the art can use many conventional methods to detect TRBC2 in the sample, and these methods are suitable for performing the second step of the diagnostic method of the present invention. Immunoassays are particularly useful. Therefore, the use of the first or second diagnostic agent of the present invention can be particularly useful for performing the diagnostic method according to the present invention. The characteristics and specific embodiments of the diagnostic agent of the present invention have been previously defined, and the same apply to the diagnostic method of the present invention.

[0509] In the diagnostic method of the present invention, a percentage of 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% or higher of TRBC2-positive T cells in the sample may indicate the presence of T cell lymphoma or leukemia.

[0510] As will be understood by those skilled in the art, the prediction, although preferably, need not be correct for 100% of the subjects to be diagnosed or evaluated. However, the term requires that a statistically significant portion of the subjects can be identified as having an increased likelihood of a given outcome. Those skilled in the art can readily determine whether the data obtained from the subjects is statistically significant using a variety of well-known statistical assessment tools, for example, using determination of confidence intervals, p-value determination, classification rates of cross-validation, etc. See Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983 for details. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The p-value is preferably 0.01 or 0.005 or less.

[0511] In addition, considering the ability of the variant antigen-binding domains or antibodies of the present invention to specifically bind to TRBC2-positive T cells, they can also be used for in vivo diagnosis of T cell lymphoma or leukemia. For example, they can be used in medical imaging, i.e., a set of techniques and methods for creating images of the body (or its parts and functions) such as the human body for clinical purposes, such as medical procedures seeking to reveal, diagnose or examine diseases.

[0512] For this purpose, the variant antigen-binding domains or antibodies of the present invention are labeled by suitable methods known in the art and provided as reagents for diagnostic imaging methods, for example, by conjugating and / or loading appropriate molecules such as radioisotopes or fluorescent dyes. Such diagnostic imaging methods include, for example, radioimmuno-diagnosis, positron emission tomography (PET), endoscopic immunofluorescence methods, etc. The variant antigen-binding domains and antibodies of the present invention can be conjugated with γ-emitting isotopes and used in radioimmunoscintigraphy or single photon emission computed tomography using a γ camera. The variant antigen-binding domains and antibodies of the present invention can be conjugated with positron emitters and used in PET. The variant antigen-binding domains and antibodies of the present invention can be conjugated with fluorescent dyes such as Cy3, Cy2, Cy5 or FITC and used in endoscopic immunofluorescence methods. The variant antigen-binding domains and antibodies of the present invention modified as described are administered by any suitable route, such as intravenously, in a dose suitable for the individual, and the location of TRBC2-positive T cells is detected, determined or measured by methods well known in the art. The methods and techniques used herein, including diagnostic imaging, are known to those skilled in the art, and those skilled in the art can also provide suitable dosage formulations.

[0513] The T-cell lymphoma or leukemia to be diagnosed may be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma, nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0514] The sample may be a blood sample or may be derived from a blood sample.

[0515] 13. Method for Personalized Medicine

[0516] In another aspect, the present invention provides a method for identifying a subject having a T-cell lymphoma or leukemia suitable for treatment with the cells, antibodies, BiTEs, or conjugates of the present invention, hereinafter referred to as " The first method for personalized medicine of the present invention First method ", which comprises determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0517] The terms "cells of the present invention", "antibodies of the present invention", "BiTEs of the present invention", "chemotherapeutic agents of the present invention", "subject", and "sample containing T cells" have been described in detail in the context of the previous aspects of the present invention, and their characteristics and embodiments are equally applicable to this aspect of the present invention.

[0518] In the first method of personalized medicine of the present invention, if the percentage of TRBC2-positive T cells in the sample is 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or higher, the subject is suitable for the treatment with the cells, antibodies, BiTEs or chemotherapeutic conjugates of the present invention.

[0519] In another aspect, the present invention provides a method for selecting a therapy comprising the cells, antibodies, BiTEs, or chemotherapeutic conjugates of the present invention for treating a subject, hereinafter referred to as " The second method for personalized medicine of the present invention ", which comprises determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0520] The terms "cells of the present invention", "antibodies of the present invention", "BiTEs of the present invention", "chemotherapeutic agents of the present invention", "subject", and "sample containing T cells" have been described in detail in the context of the previous aspects of the present invention, and their characteristics and embodiments are equally applicable to this aspect of the present invention.

[0521] In a second method of personalized medicine of the present invention, if the percentage of TRBC2-positive T cells in a sample is 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or higher, a therapy comprising a cell, an antibody, a BiTE, or a chemotherapeutic conjugate of the present invention is selected to treat the subject.

[0522] The sample can be a blood sample or can be derived from a blood sample.

[0523] The T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0524] The present invention will now be further described by way of examples, which are intended to assist a person of ordinary skill in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way. Examples

[0525] Example 1: Analysis of the Binding of Anti-TRBC2 Antibody by Surface Plasmon Resonance (SPR)

[0526] The crystal structure of the TRBC1-specific monoclonal antibody hJovi-1 was solved as a complex with TRBC1-TCR to ( Figure 3 ). Through computational biology and protein engineering, several mutant forms of the anti-TRBC1 conjugate were rationally designed to switch the specificity from TRBC1 to TRBC2. Many anti-TRBC2 conjugates were produced in the IgG form. Table 1 shows the details of the mutations included in the VH and VL domains of hJovi-1.

[0527] Using a Biacore T200 instrument, the S-series CM5 sensor chip was immobilized with anti-human Fc capture antibody by amine coupling to a density of 9000 - 10000 RU. Under all experimental conditions, HBS-P+ buffer was used as the running buffer. The tested anti-TRBC2 antibodies (Table 1) were captured in flow cells 2, 3, and 4 at a density of 100 - 300 RU. Recombinant purified TRBC1 or TRBC2 at known concentrations was used as the "analyte" and was injected into each flow cell at a flow rate of 30 μl / min with a contact time of 150 s and a dissociation time of 300 s. In each experiment, flow cell 1 was unmodified and was used for reference subtraction. The "0 concentration" sensorgram of the buffer alone was used for dual reference subtraction to account for the effect of drift. The data was fitted to a 1:1 Langmuir binding model. Since a capture system was used, local R max parameters were used for data fitting.

[0528] Table 1 shows the results of the affinity for TRBC1 and TRBC2 obtained for each anti-TRBC2 antibody. The tested anti-TRBC2 antibodies generally showed preferential binding to TRBC2 in the nM range and had different ka and kd kinetic properties. In the vast majority of cases, the binding to TRBC1 was evaluated as >1 μM, with values close to the sensitivity limit of the instrument.

[0529] Example 2: Generation of Anti-TRBC2 CAR Based on KFN Conjugate

[0530] Based on the anti-TRBC2 triple mutant (T28K, Y32F, A100N mutations in the VH domain of hJovi-1, SEQ ID NO: 26) and humanized Jovi-1 (hJovi-1), second-generation CAR constructs ( Figure 4 ) were generated. These CAR constructs were cloned into a retroviral vector and used to transduce activated PBMCs obtained from healthy donors.

[0531] Example 3: Functional Characterization of Anti-TRBC2 CAR: Cytokine Production

[0532] To evaluate the functional ability of anti-TRBC2 triple mutant CAR-T cells against TRBC2, a plate-binding assay was used, in which TRBC1 or TRBC2 was immobilized before the addition of CAR-T cells. After 72 hours, the culture supernatant was collected and the production of IFN-γ was measured by ELISA. In Figure 5 A, compared to TRBC1, anti-TRBC2 triple mutant CAR-T cells showed more IFN-γ release in the presence of the TRBC2 ligand. In contrast, hJovi-1 CAR-T cells showed high IFN-γ production only when cultured with the TRBC1 ligand ( Figure 5B). These results indicate that CAR-T cells transduced with the anti-TRBC2 triple mutant have higher activation and cytokine release against TRBC2, but not when exposed to TRBC1.

[0533] Example 4: Functional Characterization of Anti-TRBC2 CAR: Cytotoxicity Assay

[0534] To determine the ability of the anti-TRBC2 triple mutant to target TRBC2, a cytotoxicity assay was established using Raji cells transduced to express TRBC1 or TRBC2 as target cells and co-cultured with CAR-T cells. hJovi-1 CAR-T cells or anti-TRBC2 triple mutant CAR-T cells were co-cultured with Raji WT, Raji TRBC1 + or Raji TRBC2 + cells at a ratio of 1:1 (E:T). The recovery of target cells was measured by flow cytometry 72 hours after culture and used to establish the cytotoxic ability of CAR-T cells. Cultures containing anti-TRBC2 triple mutant CAR-T cells showed limited survival of Raji TRBC2 + target cells ( Figure 6 ). In contrast, anti-TRBC2 triple mutant CAR-T cells did not eliminate Raji TRBC1 + target cells, indicating their enhanced ability to target TRBC2 + cells.

[0535] We used engineered monoclonal antibodies to generate a second-generation anti-TRBC2 CAR. We demonstrated that our anti-TRBC2 CAR showed specificity, cytokine release, and cytotoxicity against TRBC2+ cell lines in 72-hour co-cultures, but not against TRBC1+ cell lines or cell lines that do not express TCR on the surface. Anti-TRBC2 CAR T cells also showed proliferative ability in long-term co-culture assays.

[0536] Example 5: Functional Characterization of Anti-TRBC2 CAR: Killing Assay

[0537] Additional second-generation CAR constructs were generated based on the anti-TRBC2 binders in Table 1 with the following mutations:

[0538] - T28K, Y32F, A100N mutations in the VH domain of hJovi-1 and N35K in the VL domain (referred to as N35K, SEQ ID NO:25),

[0539] - T28K, Y32F, A100N, N103L mutations in the VH domain of hJovi-1 (referred to as N103L, SEQ ID NO:27),

[0540] The T28K, Y32F, A100N, N103M mutations in the VH domain of -hJovi-1 and N35Y in the VL domain (referred to as N103M-N35Y, SEQ ID NO:28),

[0541] The T28K, Y32F, A100N, Y102F, N103M mutations in the VH domain of -hJovi-1 and N35R in the VL domain (referred to as Y102F-N103M-N35R, SEQ ID NO:29),

[0542] The T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain of -hJovi-1 and N35R in the VL domain (referred to as Y102L-N103M-N35R, SEQ ID NO:30).

[0543] These CAR constructs and the anti-TRBC2 triple mutant (the T28K, Y32F, A100N mutations in the VH domain of hJovi-1, referred to as KFN, SEQ ID NO:26) were cloned into a retroviral vector and used to transduce (a) Jurkat cells or (b) activated PBMCs obtained from healthy donors. In the killing assay using activated PBMCs, the anti-TRBC1 control CAR (i.e., the hJovi-1 CAR) construct was used as a control.

[0544] a) Jurkat cells

[0545] To evaluate whether the resulting conjugates were specific for the TRBC2 antigen, TRBC1+ Jurkat cells were transduced with the above anti-TRBC2 CAR constructs. HPB-ALL cells transduced to express only TRBC1 or TRBC2 (referred to as HPB TRBC1 and HPB TRBC2, respectively) were used as target cells, and co-cultures of the transduced Jurkat cells were established at an effector cell to target (E:T) ratio of 1:1, with HPB-ALL cells with knocked-out TCR (referred to as HPB KO) used as a negative control. The transduced Jurkat cells were plated separately or together with αCD3 / αCD28 antibodies and used as negative or positive assay controls, respectively. Antigen-specific activation was evaluated by flow cytometry via CD69 staining after 24 hours.

[0546] Figure 7The results shown in [Figure 0] indicate that all transduced Jurkat cells were selectively activated after co-incubation with HPB TRBC2 cells. These results suggest that all tested CARs showed specificity for the TRBC2 target, as no CD69 upregulation was observed when Jurkat was co-cultured with TRBC1+ or HPB KO targets.

[0547] b) PBMC

[0548] First, after staining with 10 ug / ml of biotinylated murine JOVI-1 antibody that binds only to TRBC1, PBMCs were separated into TRBC1+ and TRBC2+ cells using magnetic anti-biotin-coated beads. The TRBC2+ population was transduced with hJovi-1CAR (JOVI), and the TRBC1+ population was transduced with the anti-TRBC2 CAR described above. This differential transduction ensured that antigen activation and target killing were triggered only when the target was added under controlled culture conditions (e.g., effector-to-target ratio and time point), rather than in a mixed population where the conditions for each PBMC donor were different.

[0549] Second, a killing assay was established using HPB-ALL cells transduced to express only TRBC1 or TRBC2 (designated TRBC1+HPB-ALL and TRBC2+HPB-ALL, respectively) as target cells, with HPB-ALL cells with a knocked-out TCR (designated HPB-KO) as a control. Ten days after transduction, the cells were co-cultured at an E:T ratio of 1:2. Seventy-two hours after the assay was established, cell killing was evaluated by flow cytometry.

[0550] The results showed that hJovi-1 CAR-T cells killed only TRBC1+HPB-ALL cells, while all anti-TRBC2 CAR-T cells killed only TRBC2+HPB-ALL cells ( Figure 8 ). Similar background killing levels were observed for all TRBC1- and TRBC2-specific CAR-T cells on HPB-KO control cells, indicating that all tested CARs were specific for their cognate antigens.

[0551] Example 6: Analysis of the Influence of Different Antibody Forms on the Binding of Anti-TRBC2 Antibody by Surface Plasmon Resonance (SPR) Influence

[0552] The multimerization in the anti-TRBC2 conjugate was analyzed by SPR. For this purpose, anti-TRBC2 having mutations T28K, Y32F, A100N, Y102L and N103M in the VH domain and N35R mutation in the VL domain of the hJovi-1 antibody was used in three different forms, namely the scFv (SEQ ID NO: 22), scFv-Fc (SEQ ID NO: 23) and scFv-COMP (SEQ ID NO: 24) antibody forms.

[0553] SEQ ID NO:22

[0554] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGLMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKR

[0555] SEQ ID NO:23

[0556] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGLMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSDPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0557] SEQ ID NO:24

[0558] QVQLVQSGAEVKKPGASVKVSCKASGYKFTGFVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGNGLMFDGAYRFFDFWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGRTYLHWYLQKPGQSPRLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIKRSGGGGSGGGGSGGGGSAGSDLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACGSGKKDPKSGGGGSYPYDVPDYA

[0559] To test the 1:1 binding interaction of the anti-TRBC2 scFv, the S-series CM5 sensor chip was immobilized with an anti-human Fc capture antibody by amine coupling using a Biacore T200 instrument at a density of 9000 - 10000 RU. Under all experimental conditions, HBS-P+ buffer was used as the running buffer. The tested anti-TRBC2 antibody was captured on flow cell 4 to a density of 200 - 250 RU. Recombinant purified TRBC1 or TRBC2 at known concentrations was used as the "analyte" and was injected into each flow cell at a flow rate of 30 μl / min with a contact time of 150 s and a dissociation time of 300 s. Flow cell 1 was unmodified and was used for reference subtraction. The "0 concentration" sensorgram of the buffer alone was used for double reference subtraction to account for the effect of drift. The data was fitted to a 1:1 Langmuir binding model. Since a capture system was used, local R max parameters were used for data fitting in each case. The dissociation rate was determined and the half-life was calculated according to the following formula: t 1 / 2 = ln2 / kd.

[0560] Figure 9 The results shown in A indicate that the half-life of the anti-TRBC2 scFv conjugate is 8 s. The binding affinity of the conjugate is shown in Table 1.

[0561] To test the multivalent interaction of the scFv-Fc and scFv-COMP antibody forms, the S-series CAP chip was immobilized with a biotin capture reagent to 2500 - 5000 RU. In separate experimental cycles, soluble recombinant biotinylated TRBC1 and TRBC2 were captured on flow cell 3 to a density of 90 - 110 RU. The purified anti-TRBC2 scFv-Fc or scFv-COMP antibody was injected into the flow cell at a flow rate of 30 μl / min with a contact time of 150 s and a dissociation time of 300 s. On flow cell 1, the biotinylated protein was omitted and was used for reference subtraction. The "0 concentration" sensorgram of the buffer alone was used for double reference subtraction to account for the effect of drift. The data was fitted to a 1:1 Langmuir binding model. Since a capture system was used, local Rmax parameters were used for data fitting in each case. The dissociation rate was determined and the half-life was calculated according to the following formula: t 1 / 2 = ln2 / kd.

[0562] Figure 9 The results shown in B and 9C indicate that the half-lives of the scFv-Fc and scFv-COMP antibody forms are 16.2 s and 7.3 min, respectively.

[0563] Thus, these results demonstrate that multimerization improves the binding of low-affinity / high-specificity TRBC2 antibodies.

[0564] This application claims the benefit of UK Application No. 1817822.8, filed on October 31, 2018. This application is incorporated herein by reference in its entirety.

[0565] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described ways of implementing the invention that are obvious to those skilled in the molecular biology or related fields are intended to fall within the scope of the appended claims.

Claims

1. A variant antigen-binding domain, wherein the mutations of the variant antigen-binding domain compared to a reference antibody are selected from the following: - T28K, Y32F, A100N in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N in the VH domain, - T28K, Y32F, A100N, Y27N in the VH domain, - T28K, Y32F, A100N, G31K in the VH domain, - T28K, Y32F, A100N, Y27M in the VH domain, - T28K, Y32F, A100N, Y27W in the VH domain, - T28K, Y32F, A100N in the VH domain and R55K in the VL domain, - T28K, Y32F, A100N, N103H in the VH domain, - T28K, Y32F, A100N, N103A in the VH domain, - T28K, Y32F, A100N, N103Y in the VH domain, - T28K, Y32F, A100N in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103S in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain, - T28K, Y32F, A100N, N103W in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103S in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, R98K in the VH domain, - T28K, Y32F, A100N, N103S in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain, - T28K, Y32F, A100N, N103S in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103S in the VH domain and N35Y in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103W in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain and N35Y in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain, - T28K, Y32F, A100N, N103W in the VH domain, - T28K, Y32F, A100N in the VH domain, N103L and N35K in the VL domain, - T28K, Y32F, A100N in the VH domain, N103L and N35F in the VL domain, - T28K, Y32F, A100N in the VH domain, N103W and N35M in the VL domain, - T28K, Y32F, A100N in the VH domain, N103F and N35Y in the VL domain, - T28K, Y32F, A100N, Y27F in the VH domain, - T28K, Y32F, A100N, N103Q in the VH domain, - T28K, Y32F, A100N, N103S in the VH domain, - T28K, Y32F, A100N, N103M and N35F in the VL domain, - T28K, Y32F, A100N, N103F and N35M in the VL domain, - T28K, Y32F, A100N, N103F and N35F in the VL domain, - T28K, Y32F, A100N, G31R in the VH domain, - T28K, Y32F, A100N, N103W and N35F in the VL domain, - T28K, Y32F, A100N, V2R in the VH domain, - T28K, Y32F, A100N, G31S in the VH domain, - T28K, Y32F, A100N, A107S in the VH domain, - T28K, Y32F, A100N, N103E and N35M in the VL domain, - T28K, Y32F, A100N, V2K in the VH domain, - T28K, Y32F, A100N, N103E in the VH domain, - T28K, Y32F, A100N, Y102F, N103M and N35K in the VL domain, - T28K, Y32F, A100N, Y102F, N103M and N35F in the VL domain, - T28K, Y32F, A100N, Y102F, N103M and N35R in the VL domain, - T28K, Y32F, A100N, Y102F and N35R in the VL domain, - T28K, Y32F, A100N, N103M and N35M in the VL domain, - T28K, Y32F, A100N, N103M and N35Y in the VL domain, - T28K, Y32F, A100N, N103M and N35R in the VL domain, - T28K, Y32F, A100N, N103F and N35K in the VL domain, - T28K, Y32F, A100N, Y102L, N103W in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, Y102L, N103W in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, Y102F in the VH domain, - T28K, Y32F, A100N, Y102L, N103M in the VH domain and N35R in the VL domain, - G31R, Y32F, A100N in the VH domain, or - T28R, Y32F, A100N in the VH domain; wherein the reference antibody comprises a VH domain and a VL domain and the amino acid sequence of the VH domain is as shown in SEQ ID NO:1 and the amino acid sequence of the VL domain is as shown in SEQ ID NO:2; and wherein the variant antigen-binding domain shows an increased affinity for TRBC2 relative to the reference antibody.

2. An antibody comprising the variant antigen-binding domain according to claim 1.

3. A chimeric antigen receptor (CAR) comprising the variant antigen-binding domain according to claim 1, a spacer, a transmembrane domain, and an intracellular domain.

4. A bispecific T cell engager comprising the variant antigen-binding domain according to claim 1 and a T cell activation domain.

5. A nucleic acid sequence encoding the variant antigen-binding domain according to claim 1, the antibody according to claim 2, the CAR according to claim 3, or the bispecific T cell engager according to claim 4.

6. A vector comprising the nucleic acid sequence according to claim 5.

7. A cell comprising the CAR according to claim 3.

8. A method for preparing the cell according to claim 7, comprising the step of transducing or transfecting a cell with the vector according to claim 6 comprising a nucleic acid sequence encoding the CAR.

9. A conjugate comprising the variant antigen-binding domain according to claim 1 or the antibody according to claim 2, and a detectable entity or a chemotherapeutic entity.

10. Use of the cell according to claim 7, or the antibody according to claim 2, or the bispecific T cell engager according to claim 4, or the conjugate according to claim 9 in the preparation of a medicament for the treatment of T cell lymphoma or leukemia, wherein malignant T cells express TRBC2.

11. Use of the variant antigen-binding domain according to claim 1 or the antibody according to claim 2 in the preparation of a kit for the diagnosis of T cell lymphoma or leukemia in a subject.

12. The use according to claim 11, wherein the kit is used in a method comprising the step of contacting the variant antigen-binding domain according to claim 1 or the antibody according to claim 2 with a sample comprising T cells from the subject.

13. Use of a reagent for determining the percentage of TRBC2-positive T cells in a T cell-containing sample from a subject in the preparation of a kit for identifying a subject with T cell lymphoma or leukemia suitable for treatment with the cell according to claim 7, or the antibody according to claim 2, or the bispecific T cell engager according to claim 4, or the conjugate according to claim 9.

14. Use of a reagent for determining the percentage of TRBC2-positive T cells in a T cell-containing sample from a subject in the preparation of a kit for selecting a subject with T cell lymphoma or leukemia for treatment with a therapy comprising the cell according to claim 7, or the antibody according to claim 2, or the bispecific T cell engager according to claim 4, or the conjugate according to claim 9.

15. Use according to any one of claims 10 to 14, wherein the T cell lymphoma or leukemia is selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

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