Joint domain

Abstract

Provision of combined domains. [Solution] The present invention provides a variant antigen-binding domain comprising at least one mutation in the VH domain compared to a reference antibody, exhibiting increased affinity to TRBC2 compared to the reference antibody. It further provides an antibody, a chimeric antigen receptor (CAR), and a bispecific T cell engager (BiTE), a cell containing the CAR, and a conjugate containing the variant antigen-binding domain or the antibody. Furthermore, it provides medical uses, diagnostic methods, and personalized drug methods that utilize the products of the present invention.

Description

[Technical Field]

[0001] Field of Invention This invention relates to a variant antigen-binding domain that specifically binds to TRBC2. It also relates to cells and agents that are beneficial in the treatment and diagnosis of T-cell malignancies. [Background technology]

[0002] Background of the Invention Lymphoid malignancies can be broadly divided into those originating from T cells or those originating from B cells. T-cell malignancies are a group of clinically and biologically heterogeneous disorders, together accounting for 10-20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histological subtypes are peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; and angioimmunoblastic T-cell lymphoma. These include peritoneal lymphoma (AITL) and anaplastic large cell lymphoma (ALCL). Approximately 20% of all acute lymphoblastic leukemias (ALL) exhibit a T-cell phenotype.

[0003] These conditions generally behave more aggressively than, for example, B-cell malignancies, with an estimated 5-year survival rate of only 30%. In the case of T-cell lymphoma, a high proportion of patients exhibit disseminated disease, unfavorable International Prognostic Indicator (IPI) scores, and a high incidence of extranodal disease. Chemotherapy alone is usually ineffective, and less than 30% of patients are cured with current treatments.

[0004] Furthermore, unlike B-cell malignancies, which see dramatically improved outcomes with immunotherapies such as the anti-CD20 monoclonal antibody rituximab, there are currently no equally effective and minimally toxic immunotherapies available to treat T-cell malignancies. A significant challenge in developing immunotherapies for T-cell damage is the considerable overlap in marker expression between cloned T cells and normal T cells, and the lack of a single antigen that clearly allows for the identification of cloned (malignant) cells.

[0005] Chimeric antigen receptor (CAR) T cells have shown promise in treating refractory B-cell malignancies. While targeting T-cell malignancies appears equally effective, the application of CARs in diseases such as T-cell lymphoma is hindered by a lack of suitable target antigens. Unlike B-cell lymphoma, where ablation of the B-cell compartment is manageable toxicity and can be treated with intravenous immunoglobulin administration, disrupting the T-cell compartment is less tolerable and leads to complications associated with suppression of cell-mediated immunity.

[0006] A method for treating T-cell lymphomas and leukemias consisting of targeting the constant region of the TCR beta chain (TRBC) was previously described in WO2015 / 132598. This approach is based on the unique characteristics of the T cell receptor, namely that each TCR exclusively encodes either TRBC1 or TRBC2. Since T-cell lymphomas and leukemias are clonal populations of cells, each lymphoma expresses one TCR having either TRBC1 or TRBC2 on its surface.

[0007] The monoclonal antibody Jovi-1 specifically binds to TRBC1 and is used as a CAR-binding domain for therapies treating T-cell lymphoma (Maciocia et al., 2017, Nat Med 23:1416-23;WO2015 / 132598). This proposed therapy would enable treatment of a subset of patients expressing TCRs with a constant TRBC1 region.

[0008] To treat the entire patient population, TRBC2-targeting conjugates / CARs are needed. One method for obtaining TRBC2-specific antibodies is by phage selection on a human phage-presenting library. Another method consists of immunizing animals with TRBC2-derived peptides and then selecting specific antibodies. Both approaches have been successful, and a variety of TRBC2-specific conjugates have been generated, as disclosed in WO2015 / 132598.

[0009] The present invention provides an alternative conjugate that is specific to TRBC2 and is a potential therapeutic agent for the treatment of TRBC2+ lymphoma or leukemia. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2015 / 132598 [Non-patent literature]

[0011] [Non-Patent Document 1] Maciocia et al., 2017, Nat Med 23:1416-23 [Overview of the Initiative] [Means for solving the problem]

[0012] Summary of the Invention The inventors have elucidated the crystal structure of JOVI-1, a TRBC1-specific monoclonal antibody that forms a complex with TRBC1-TCR, down to 2.4 Å (Viney et al., 1992). Hybridoma 11:701-13)(Figure 3). From the crystal structure, it was possible to engineer the original Jovi-1 antibody to bind to TRBC2. This method is particularly attractive because some of the amino acids in the antibody form a paratope that provides the shape complementarity for TCR engagement, and only a small number are required for specificity to TRBC1. Through computational biology and protein engineering, the inventors rationally designed a mutant version 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 a variant antigen-binding domain comprising at least one mutation in the VH domain compared to a reference antibody having a VH domain with the sequence shown in SEQ ID NO:1 and a VL domain with 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 the variant antigen-binding domain exhibits increased affinity for TRBC2 compared to the reference antibody.

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

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

[0016] The variant antigen-binding domain can further comprise at least one mutation at a position selected from the group consisting of 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 can be selected from the following: a) In the VH domain - V2K, V2R, - Y27F, Y27M, Y27N, Y27W, - G31K, G31R, G31S, - R98K, - Y102F, Y102L, - N103A, N103E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, N103Y, - A107S, and b) In the VL domain - N35M, N35F, N35Y, N35K, N35R, and - R55K.

[0017] The variant antigen-binding domain can be selected from variant antigen-binding domains comprising the following combinations of mutations: - 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, N103L in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103W in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain 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 in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, G31R in the VH domain, - T28K, Y32F, A100N, N103W in the VH domain 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 in the VH domain 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 in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, Y102F in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35Y in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain 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, and - T28K, Y32F, A100N, Y102L, N103M in the VH domain and N35R in the VL domain.

[0018] The variant antigen-binding domain can include T28K, Y32F, and A100N mutations in the VH domain and N35K mutations in the VL domain.

[0019] The variant antigen-binding domain can contain T28K, Y32F, and A100N mutations in the VH domain.

[0020] The variant antigen-binding domain can further demonstrate a reduced affinity for TRBC1 compared to the reference antibody.

[0021] The affinity ratio of the variant antigen-binding domains to TRBC2 and TRBC1 may be at least 2.

[0022] The affinity ratio of the variant antigen-binding domains to TRBC2 and TRBC1 may be at least 5.

[0023] The affinity ratio of the variant antigen-binding domains to TRBC2 and TRBC1 may be at least 10.

[0024] The variant antigen-binding domain may further contain an oligomerized domain.

[0025] In a second aspect, the present invention provides an antibody comprising a variant antigen-binding domain according to a first aspect of the present invention.

[0026] In a third aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a variant antigen-binding domain, a spacer, a transmembrane domain, and an endodomain according to a first aspect of the present invention.

[0027] The spacer can be selected from the human CD8 stalk shown in Sequence ID No. 7 and the COMP spacer shown in Sequence ID No. 19.

[0028] In a fourth aspect, the present invention provides a bispecific T cell engager (BiTE) comprising a variant antigen-binding domain and a T cell activation domain according to a first aspect of the present invention.

[0029] In a fifth aspect, the present invention provides a nucleic acid sequence encoding a variant antigen-binding domain according to a first aspect of the present invention, an antibody according to a second aspect of the present invention, a CAR according to a third aspect of the present invention, or a BiTE according to a fourth aspect of the present invention.

[0030] In a sixth aspect, the present invention provides a vector comprising a nucleic acid sequence according to a fifth aspect of the present invention.

[0031] In a seventh aspect, the present invention provides cells containing CARs according to a third aspect of the present invention.

[0032] In an eighth aspect, the present invention provides a method for producing cells according to a seventh aspect of the present invention, comprising the step of transfecting or transfecting cells with a vector according to a sixth aspect of the present invention comprising a nucleic acid sequence encoding a CAR.

[0033] In a ninth aspect, the present invention provides a conjugate comprising a variant antigen-binding domain according to a first aspect of the present invention or an antibody according to a second aspect of the present invention and a detectable entity or a chemotherapeutic entity.

[0034] The conjugate may contain a chemotherapeutic entity.

[0035] In a tenth aspect, the present invention provides a method for treating a T-cell lymphoma or leukemia in a subject, comprising the step of administering cells according to aspect 7 of the present invention, or an antibody according to aspect 2 of the present invention, or a BiTE according to aspect 4 of the present invention, or a conjugate according to aspect 9 of the present invention, to the subject, wherein the malignant T cells express TRBC2.

[0036] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0037] In an eleventh aspect, the present invention provides cells according to the seventh aspect of the present invention, or antibodies according to the second aspect of the present invention, or BiTE according to the fourth aspect of the present invention, or conjugates according to the ninth aspect of the present invention, for use in pharmaceuticals.

[0038] In a twelfth aspect, the present invention provides cells according to the seventh aspect of the present invention, an antibody according to the second aspect of the present invention, 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.

[0039] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0040] In a thirteenth aspect, the present invention provides the use of cells according to aspect seven of the present invention, or antibodies according to aspect two of the present invention, or BiTE according to aspect four of the present invention, or conjugates according to aspect nin of the present invention, in the manufacture of a pharmacopoeia for treating T-cell lymphoma or leukemia, wherein malignant T cells express TRBC2.

[0041] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0042] In a fourteenth aspect, the present invention provides a diagnostic agent comprising a variant antigen-binding domain according to a first aspect of the present invention or an antibody according to a second aspect of the present invention.

[0043] The diagnostic agent may be used to diagnose T-cell lymphoma or leukemia.

[0044] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0045] In a fifteenth aspect, the present invention provides a method for diagnosing T-cell lymphoma or leukemia in a subject, comprising the step of contacting a sample containing T cells from the subject with a variant antigen-binding domain according to a first aspect of the present invention or an antibody according to a second aspect of the present invention.

[0046] A method for diagnosing T-cell lymphoma or leukemia in a subject may further include the step of determining the percentage of TRBC2-positive T cells in the sample.

[0047] A percentage of TRBC2-positive T cells in a sample of 70% or higher may indicate the presence of T-cell lymphoma or leukemia.

[0048] The sample may be a blood sample or derived from one.

[0049] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0050] In a sixteenth aspect, the present invention provides a method for identifying subjects of T-cell lymphoma or leukemia who are eligible for treatment with cells according to aspect 7 of the present invention, or antibodies according to aspect 2 of the present invention, or BiTE according to aspect 4 of the present invention, or conjugates according to aspect 9 of the present invention, the method comprising the step of determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0051] Subjects may be eligible for the treatment with cells according to the 7th aspect of the present invention, antibodies according to the 2nd aspect of the present invention, BiTE according to the 4th aspect of the present invention, or conjugates according to the 9th aspect of the present invention if the percentage of TRBC2-positive T cells in the sample is 70% or higher.

[0052] The sample may be a blood sample or derived from one.

[0053] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0054] In a 17th aspect, the present invention provides a method for selecting a therapy for treatment of a subject, comprising cells according to aspect 7 of the present invention, or antibodies according to aspect 2 of the present invention, or BiTE according to aspect 4 of the present invention, or a conjugate according to aspect 9 of the present invention, the method comprising the step of determining the percentage of TRBC2-positive T cells in a sample comprising T cells from a subject.

[0055] The aforementioned therapy may be selected to treat the subject if the percentage of TRBC2-positive T cells in the sample is 70% or higher.

[0056] The sample may be a blood sample or derived from one.

[0057] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia. [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 shows the αβ T cell receptor / CD3 complex. The T cell receptor is formed from six different protein chains that must be assembled in the endoplasmic reticulum in order to be expressed on the cell surface. The four proteins of the CD3 complex (CD3ζ, CD3γ, CD3ε, and CD3δ) enclose the T cell receptor (TCR). This TCR imparts specific antigenic specificity to the complex and consists of two chains: TCRα and TCRβ. Each TCR chain has a variable component distal to the membrane and a constant component proximal to the membrane. Almost all T cell lymphomas and many T cell leukemias express the TCR / CD3 complex. [Figure 2]Figure 2 shows the separation of T cell receptor β-constant region (TRBC)-1 and TRBC2 during T cell receptor rearrangement. Each TCR β-chain is formed from genomic recombination of a specific β-variable (V), diversity (D), linkage (J), and constant (TRBC) region. The human genome contains two very similar, functionally equivalent TRBC loci, known as TRBC1 and TRBC2. During TCR gene rearrangement, the J region rejoins with either TRBC1 or TRBC2. This rearrangement is permanent. Since T cells express many copies of a single TCR on their surface, each T cell expresses a TCR in which its β-chain constant region is encoded by either TRBC1 or TRBC2. [Figure 3] Figure 3 shows the structural interface between the TCR beta and Fab fragment of the TRBC1-specific antibody Jovi-1. [Figure 4] Figure 4 shows the structures of TRBC1 and TRBC2-specific chimeric antigen receptors (CARs). [Figure 5] Figure 5 shows the functional characterization of anti-TRBC2 CARs. IFN-γ generation by (A) anti-TRBC2 triple mutant CARs and (B) anti-TRBC1 CARs incubated in the presence of TRBC1 or TRBC2. [Figure 6] Figure 6 shows the 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. [Figure 7]Figure 7 shows antigen-specific activation of Jurkat cells transduced with anti-TRBC2 CAR 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 in a 1:1 E:T ratio. Controls included untransduced Jurkat cells (NT), HPB-ALL cells with knockout TCRs (referred to as HPB KO), and transduced Jurkat cells plated alone as a negative control, or with αCD3 / αCD28 antibodies as a positive assay control. N35K: A binder containing the T28K, Y32F, and A100N mutations in the VH domain of hJovi-1 and the N35K mutation in the VL domain; N103L: A binder containing the T28K, Y32F, A100N, and N103L mutations in the VH domain of hJovi-1; N103M-N35Y: A binder containing the T28K, Y32F, A100N, and N103M mutations in the VH domain of hJovi-1 and the N35Y mutation in the VL domain. A binder having the following mutations: Y102F-N103M-N35R: a binder having the T28K, Y32F, A100N, Y102F, N103M mutations in the VH domain of hJovi-1 and the N35R mutation in the VL domain; and Y102L-N103M-N35R: a binder having the T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain of hJovi-1 and the N35R mutation in the VL domain. [Figure 8]Figure 8 shows the 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. Transduced PBMCs were co-incubated with TRBC1+HPB-ALL and TRBC2+HPB-ALL cells in a 1:2 E:T ratio. Controls included untransduced cells (NT), cells transduced with anti-TRBC1hJovi-1CAR (JOVI), and HPB-ALL cells with knocked-out TCRs (HPB-KO). N35K: A binder containing the T28K, Y32F, and A100N mutations in the VH domain of hJovi-1 and the N35K mutation in the VL domain; N103L: A binder containing the T28K, Y32F, A100N, and N103L mutations in the VH domain of hJovi-1; N103M-N35Y: A binder containing the T28K, Y32F, A100N, and N103M mutations in the VH domain of hJovi-1 and the N35Y mutation in the VL domain. A binder having the following mutations: Y102F-N103M-N35R: a binder having the T28K, Y32F, A100N, Y102F, N103M mutations in the VH domain of hJovi-1 and the N35R mutation in the VL domain; and Y102L-N103M-N35R: a binder having the T28K, Y32F, A100N, Y102L, N103M mutations in the VH domain of hJovi-1 and the N35R mutation in the VL domain. [Figure 9A] Figure 9 shows an analysis of the effects of different antibody formats on the binding of anti-TRBC2 antibodies by surface plasmon resonance (SPR). [Figure 9B] Figure 9 shows an analysis of the effects of different antibody formats on the binding of anti-TRBC2 antibodies by surface plasmon resonance (SPR). [Figure 9C] Figure 9 shows an analysis of the effects of different antibody formats on the binding of anti-TRBC2 antibodies by surface plasmon resonance (SPR). [Modes for carrying out the invention]

[0059] Detailed description of the invention The inventors elucidated the crystal structure of Jovi-1, which helped identify two key residues essential for TRBC1 specificity (Figure 1). Interestingly, these residues are located in CDR1, in contrast to CDR3, which typically promotes antibody specificity. Furthermore, other residues located in close proximity to the TRBC1 epitope are likely essential for manipulating TRBC1-to-TRBC2 specificity. The residues in Jovi-1 that are involved in TRBC1 binding or are important for generating TRBC2 specificity are described herein.

[0060] The present invention provides a variant of the antigen-binding domain of JOVI-1 that has a higher affinity for the TCR beta constant region 2 (TRBC2) than JOVI-1.

[0061] 1. TCRβ constant region (TRBC) T cell receptors (TCRs) are expressed on the surface of T lymphocytes and play a role in recognizing antigens bound to major histocompatibility complex (MHC) molecules. When TCRs engage with antigen peptides and MHC (peptide / MHC), T lymphocytes are activated through a series of biochemical events mediated by related enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

[0062] The TCR is a disulfide-linked, membrane-bound heterodimer consisting of highly variable alpha (α) and beta (β) chains, typically expressed as part of a complex with an immutable CD3 chain molecule. T cells expressing this receptor are called α:β (or αβ) T cells (approximately 95% of total T cells). A small number of T cells express an alternative receptor formed by variable gamma (γ) and delta (δ) chains, and these are called γδ T cells (approximately 5% of total T cells).

[0063] Each α and β chain consists of two extracellular domains: a variable (V) region and a constant (C) region, both of which are immunoglobulin superfamily (IgSF) domains that form an antiparallel β-sheet. The constant region is proximal to the cell membrane, with a transmembrane region and a short cytoplasmic tail behind it, while the variable region binds to the peptide / MHC complex. The constant region of the TCR consists of a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby creating a link between the two chains.

[0064] Both the α and β chain variable domains of the TCR possess three hypervariable or complementarity-determining regions (CDRs). The β chain variable region also possesses additional hypervariability (HV4), but this region does not typically come into contact with the antigen and is therefore not considered a CDR.

[0065] The TCR also contains up to five invariant chains γ, δ, ε (collectively referred to as CD3) and ζ. The CD3 and ζ subunits mediate TCR signaling through specific cytoplasmic domains that interact with second messenger and adapter molecules after antigen recognition by αβ or γδ. Prior to cell surface expression of the TCR complex, the subunits assemble in pairs, where both the transmembrane and extracellular domains of TCR α and β, as well as CD3 γ and δ, play their roles.

[0066] Therefore, the TCR is generally composed of the CD3 complex and the TCR α and β chains, which in turn are composed of a variable region and a constant region (Figure 1).

[0067] The locus (Chr7:q34) that supplies the TCRβ constant region (TRBC) has doubled over evolutionary history, giving rise to two nearly identical and functionally equivalent genes: TRBC1 and TRBC2 (Figure 2). Each TCR exclusively contains either TRBC1 or TRBC2, and thus each αβT cell exclusively expresses either TRBC1 or TRBC2.

[0068] The inventors previously determined that, despite the similarity between the sequences of TRBC1 and TRBC2, it is possible to distinguish them. The inventors 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).

[0069] 2. Variant antigen-binding domain In a first aspect, the present invention provides a variant antigen-binding domain, hereafter referred to as "the variant antigen-binding domain of the present invention," which includes at least one mutation in the VH domain compared to a reference antibody having 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 the at least one mutation in the VH domain is selected from T28K, Y32F, and A100N, and the variant antigen-binding domain exhibits increased affinity to TRBC2 compared to the reference antibody.

[0070] As used herein, the terms “variant” or “mutant” refer to polypeptides specifically listed, i.e., polypeptides that differ from a reference or parent polypeptide by the insertion, deletion, and / or substitution of amino acids, for example, using recombinant DNA technology or by novel synthesis. Variants and mutants are used interchangeably in connection with the present invention. The variant antigen-binding domains of the present invention include antigen-binding molecules in which one or more amino acid residues are modified by substitution, addition, and / or deletion in a manner that substantially affects the antigen-binding affinity of the reference or parent antigen-binding domain.

[0071] As used herein, the term "antigen-binding domain" refers to the variable regions of the light and heavy chains of an antibody, namely the VL and VH domains, respectively, that form the binding site. They are characterized by the same general structure, which consists of a relatively conserved region called a framework (FR) that links to three hypervariable regions called complementarity-determining regions (CDRs) (Kabat et al., 1991, Sequences of Proteins of Immunological). Interest, 5 th Ed., NIH Publication No. 91-3242, Bethesda, MD.;Chothia (& Lesk, 1987, J Mol Biol 196:901-17). The term "complementarity" as used herein. A "Determining Region" or "CDR" refers to a region within an antibody that captures the shape of an antigen. Therefore, the CDR determines the protein's affinity (roughly speaking, its binding strength) and specificity to a specific antigen. The CDRs of the two strands in each pair are aligned by a framework region, acquiring the ability to bind to a specific epitope.

[0072] The variant antigen-binding domain of the present invention contains at least one mutation in the VH domain compared to the reference antibody. 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. Mouse JOVI-1 was previously disclosed and is commercially available by Viney et al. (1992; above) (Abcam, ab5465). It has been previously determined that JOVI-1 can identify cells based on the specific expression of TRBC1 or TRBC2 by specifically binding to TRBC1 only (WO2015 / 132598). [ka]

[0073] Hereafter, unless otherwise specified, any reference to the VH domain refers to the VH domain of hJOVI-1 as shown in Sequence ID No. 1, and unless otherwise specified, any reference to the VL domain refers to the VL domain of hJOVI-1 as shown in Sequence ID No. 2.

[0074] The inventors determined that the presence of at least one mutation selected from T28K, Y32F, and A100N in the VH domain of the reference antibody induces an increased affinity of the variant antigen-binding domain of the present invention to TRBC2 compared to the reference antibody (Example 1).

[0075] As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and its epitope. High-affinity antibodies bind to a larger amount of antigen in a shorter time than low-affinity antibodies. Affinity is the k between the antibody and the antigen. off / k on The equilibrium dissociation constant (K) is the ratio of the two. D The affinity of an antigen-binding domain to any given antigen can be quantified using any conventional method, without limitation, including label-dependent methods such as direct and indirect ELISA and radioimmunoassay methods, as well as label-free methods that enable real-time direct detection and measurement of interactions such as surface plasmon resonance and biolayer interference.

[0076] The affinity of the variant antigen-binding domain of the present invention to TRBC2 is increased compared to that of the reference antibody. The affinity of the variant antigen-binding domain to 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 80%, at least 80%, at least 90%, at least 1,000%, at least 5,000%, or at least 10,000% compared to the affinity of the reference antibody to TRBC2.

[0077] The variant antigen-binding domain of the present invention may contain a T28K mutation in the VH domain. The variant antigen-binding domain of the present invention may contain a Y32F mutation in the VH domain. The variant antigen-binding domain of the present invention may contain an A100N mutation in the VH domain.

[0078] The variant antigen-binding domain of the present invention may contain at least two mutations selected from T28K, Y32F, and A100N in the VH domain. The at least two mutations may be Y32F and A100N. In one embodiment, the variant antigen-binding domain of the present invention may further contain mutations Y32F and A100N and mutation T28R in the VH domain. In another embodiment, the variant antigen-binding domain of the present invention may further contain mutations Y32F and A100N and mutation G31R in the VH domain.

[0079] The variant antigen-binding domain of the present invention may contain mutations T28K, Y32F, and A100N in the VH domain.

[0080] In another embodiment, the variant antigen-binding domain of the present invention comprises the mutations T28K, Y32F, and A100N in the VH domain, and further comprises at least one mutation at a position selected from the group consisting of V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain, and N35 and R55 in the VL domain. The variant antigen-binding domain of the present invention may contain one, two, three, four, five, six, or seven mutations at a position selected from the group consisting of V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain, and N35 and R55 in the VL domain.

[0081] The variant antigen-binding domain of the present invention may contain mutations T28K, Y32F, and A100N in the VH domain, and may further contain mutations at position V2 in the VH domain, mutations at position Y27 in the VH domain, mutations at position G31 in the VH domain, mutations at position R98 in the VH domain, mutations at position Y102 in the VH domain, mutations at position N103 in the VH domain, mutations at position A107 in the VH domain, mutations at position N35 in the VL domain, and mutations at position R55 in the VL domain.

[0082] The variant antigen-binding domain of the present invention may contain mutations T28K, Y32F, and A100N in the VH domain, and further comprises at least one mutation selected from the following: a) In the VH domain - V2K, V2R, - Y27F, Y27M, Y27N, Y27W, - G31K, G31R, G31S, - R98K, - Y102F, Y102L, - N103A, N103E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, N103Y, and - A107S, Furthermore b) In the VL domain - N35M, N35F, N35Y, N35K, N35R, and - R55K.

[0083] The variant antigen-binding domain of the present invention can be selected from variant antigen-binding domains including the following combinations of mutations: - 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, - T28K, Y32F, A100N in the VH domain and R55K in the VL domain, - T28K, Y32F, A100N in the VH domain and N35K 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, N103L in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, N103L in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103W in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain 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 in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, G31R in the VH domain, - T28K, Y32F, A100N, N103W in the VH domain 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 in the VH domain 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 in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, Y102F in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35Y in the VL domain, - T28K, Y32F, A100N, N103M in the VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain 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, and - T28K, Y32F, A100N, Y102L, N103M in the VH domain and N35R in the VL domain.

[0084] These specific combination mutations have been shown to alter binding to TRBC2 and TRBC1 in a way that is beneficial for TRBC2 targeting (see Table 1). [Table 1-1] [Table 1-2] [Table 1-3]

[0085] In certain embodiments, the variant antigen-binding domain of the present invention contains the T28K, Y32F, and A100N mutations in the VH domain.

[0086] In another specific embodiment, the variant antigen-binding domain of the present invention includes the T28K, Y32F, A100N, and Y27N mutations in the VH domain.

[0087] In another specific embodiment, the variant antigen-binding domain of the present invention includes the T28K, Y32F, A100N, and N103M mutations in the VH domain.

[0088] In another specific embodiment, the variant antigen-binding domain of the present invention includes the mutations T28K, Y32F, and A100N in the VH domain and N35K in the VL domain.

[0089] In another specific embodiment, the variant antigen-binding domain of the present invention contains the T28K, Y32F, A100N, and N103L mutations in the VH domain.

[0090] In another specific embodiment, the variant antigen-binding domain of the present invention includes the mutations T28K, Y32F, A100N, and N103M in the VH domain and the mutation N35Y in the VL domain.

[0091] In another specific embodiment, the variant antigen-binding domain of the present invention includes mutations T28K, Y32F, A100N, Y102F, and N103M in the VH domain and a mutation N35R in the VL domain.

[0092] In another specific embodiment, the variant antigen-binding domain of the present invention includes mutations T28K, Y32F, A100N, Y102L, and N103M in the VH domain and a mutation N35R in the VL domain.

[0093] It would be particularly advantageous that the variant antigen-binding domain of the present invention not only exhibits increased affinity to TRBC2 compared to the reference antibody, but also shows decreased affinity to TRBC1 compared to that of the reference antibody. This change in antigen specificity would allow the variant antigen-binding domain to distinguish between TRBC2 and TRBC1 by exhibiting preferential binding to TRBC2. Thus, in another embodiment, the variant antigen-binding domain further exhibits reduced affinity to TRBC1 compared to the reference antibody.

[0094] The affinity of the variant antigen-binding domain of the present invention to TRBC1 is lower than that of the reference antibody. The affinity of the variant antigen-binding domain to 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 80%, at least 80%, at least 90%, at least 1,000%, at least 5,000%, or at least 10,000% compared to the affinity of the reference antibody to TRBC1.

[0095] The affinity ratio of the variant antigen-binding domain of the present invention to TRBC2 and TRBC1 may 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 at least 1,000, or greater than that.

[0096] In Example 6, by increasing the binding affinity of the variant antigen-binding domain of the present invention, the inventors unexpectedly discovered that not only did the binding affinity of the variant antigen-binding domain of the present invention to TRBC2 increase, but its low affinity to TRBC1 was maintained, resulting in a dramatic improvement in the specificity of the variant antigen-binding domain of the present invention to TRBC2.

[0097] The binding affinity of the variant antigen-binding domain of the present invention to TRBC2 can be increased by using a domain having the ability to form oligomers or multimers. Therefore, the variant antigen-binding domain of the present invention may further include an oligomeric domain. As used herein, the term “oligomeric domain” refers to a domain that self-associates to form an oligomer, such as a dimer or trimer or multimer. Therefore, as used herein, the term “oligomeric domain” also refers to a multimerizing domain. Oligomerized domains are well known to those skilled in the art, and any oligomeric domain can be used with the variant antigen-binding domain of the present invention if the resulting oligomer maintains or improves the affinity of the monomeric variant antigen-binding domain to TRBC2. Examples of oligomeric domains include, but are not limited to, the Fc region, the COMP spacer of SEQ ID NO: 18, or truncated COMP, as described below in relation to the chimeric antigen receptor (CAR) of the present invention.

[0098] The present invention also intends to include different formats for the variant antigen-binding domain of the present invention, including, but not limited to, scFv, diabody, trimmerbody, minibody, F(ab) and F(ab')2 fragments, as well as complete antibodies, i.e., IgG, IgM, IgA, IgD, and IgE.

[0099] Therefore, another aspect of the present invention relates to an antibody comprising the variant antigen-binding domain of the present invention, hereafter referred to as "the antibody of the present invention."

[0100] 3. Chimeric antigen receptor In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a variant antigen-binding domain, a transmembrane domain, and an endodomain, hereafter referred to as "the CAR of the present invention."

[0101] The term “variant antigen-binding domain of the present invention” is described in detail in relation to the first aspect of the present invention, and its features and embodiments are equally applicable to this aspect of the present invention.

[0102] As used herein, the terms “chimeric antigen receptor,” “CAR,” “chimeric T cell receptor,” “artificial T cell receptor,” or “chimeric immune receptor” refer to a chimeric type I transmembrane protein that ligates an extracellular antigen-recognition domain (conjugate) to an intracellular signaling domain (endodomain). The conjugate is generally a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats that include an antigen-binding site. A spacer domain is usually required to separate the conjugate from the membrane and allow it to be oriented appropriately. A common spacer domain used is the Fc of IgG1. More compact spacers are also sufficient, for example, the stalk from CD8α, and depending on the antigen, the IgG1 hinge alone may suffice. The transmembrane domain anchors the protein within the cell membrane and ligates the spacer to the endodomain.

[0103] Early CAR designs had endodomains 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 induce T cell killing on cognitive target cells, but could not fully activate T cells to proliferate and survive. To overcome this limitation, complex endodomains were constructed: the fusion of the intracellular portion of the T cell costimulatory molecule with that of CD3ζ results in second-generation receptors capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which delivers the most potent costimulatory signal—i.e., immune signal 2 that induces T cell proliferation. Several receptors have also been described, including TNF receptor family endodomains, such as the closely related OX40 and 4-1BB, which transmit survival signals. Even more potent third-generation CARs, possessing endodomains capable of transmitting activation, proliferation, and survival signals, have now been described.

[0104] When a CAR binds to a target antigen, this results in the transmission of an activation signal to T cells that express it. Thus, CARs direct the specificity and cytotoxicity of T cells towards tumor cells expressing the target antigen.

[0105] Therefore, CARs generally include: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain including or associating with a signaling domain (see Figure 4).

[0106] CAR can have the following general structure: Antigen-binding domain - spacer domain - transmembrane domain - intracellular signal transduction domain (endodomain).

[0107] 3.1. Signal Peptides The CAR of the present invention may contain a signal peptide such that, when the CAR is expressed in cells such as T cells, the developing protein is guided to the endoplasmic reticulum and then to the cell surface where it is subsequently expressed.

[0108] The core of a signal peptide can contain a long sequence of hydrophobic amino acids that tend to form a single alpha-helix. A signal peptide can begin with a short, positively charged sequence of amino acids, which helps enforce the correct topology of the polypeptide during rearrangement. The end of a signal peptide typically contains a sequence of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after rearrangement to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0109] Signal peptides can be located at the amino terminus of a molecule.

[0110] The signal peptide may include SEQ ID NOs: 3-5, or variants 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 inducing the cell surface expression of the protein. Sequence ID 3:MGTSLLCWMALCLLGADHADG

[0111] The signal peptide of SEQ ID NO: 3 is compact and highly efficient. It is predicted to give approximately 95% cleavage behind the terminal glycine, allowing for efficient removal by signal peptidases. Sequence ID 4: MSLPVTALLLPLALLLHAARP

[0112] The signal peptide in SEQ ID NO: 4 is derived from IgG1. Sequence ID 5:MAVPTQVLGLLLLWLTDARC

[0113] The signal peptide in SEQ ID NO: 5 is derived from CD8.

[0114] 3.2. Spacer Domain CARs include a spacer sequence that ligates the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0115] In the CAR of the present invention, the spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge, or a human CD8 stalk or a mouse CD8 stalk. The spacer may instead include an alternative linker sequence, an IgG1 hinge, or a CD8 stalk having similar length and / or domain spacing characteristics to the IgG1 Fc region. The human IgG1 spacer may be modified to remove the Fc binding motif. The spacer may include a coiled-coil domain, as described, for example, in WO2016 / 151315.

[0116] The CAR of the present invention may include sequences selected from the sequences shown in SEQ ID NOs: 6 to 10 or variants thereof having at least 80% sequence identity. [ka] [ka]

[0117] It is possible to truncate the COMP coiled-coil domain at the N-terminus and retain surface expression. Therefore, a coiled-coil COMP spacer can contain or consist of a truncated version of SEQ ID NO: 19, which is truncated at the N-terminus. The truncated COMP can contain the five C-terminal amino acids of SEQ ID NO: 19, i.e., sequence CDACG (SEQ ID NO: 20). The truncated COMP can contain 5 to 44 amino acids, e.g., 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 cysteine ​​residues involved in multimerization. The truncated COMP can retain the ability to form multimers.

[0118] 3.3. Transmembrane domains A transmembrane domain is a sequence of CARs that spans across the membrane.

[0119] The transmembrane domain may be any protein structure that is thermodynamically stable within the membrane. This is generally an alpha-helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the present invention. The presence and span of the protein's transmembrane domain can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, given that protein transmembrane domains are relatively simple structures—namely, polypeptide sequences that are expected to form a hydrophobic alpha-helix long enough to span the membrane—artificially designed TM domains can also be used (U.S. Patent No. 7052906B1 describes synthetic transmembrane components).

[0120] The transmembrane domain can be derived from CD28, CD8a, or TYRP-1, which provides excellent receptor stability.

[0121] In one embodiment, the transmembrane domain originates from CD8a. Sequence ID 11: CD8a transmembrane domain IYIWAPLAGTCGVLLLSLVIT

[0122] In another embodiment, the transmembrane domain originates from TYRP-1. Sequence ID 12: TYRP-1 transmembrane domain IIAIAVVGALLLVALIFGTASYLI

[0123] 3.4. End Domain The endodomain is the signaling portion of a CAR. After antigen recognition, the receptor cluster, native CD45 and CD148, is eliminated from the synapse, and the signal is transmitted to the cell. The most commonly used endodomain component is that of CD3ζ, which contains three ITAMs. This transmits an activation signal to T cells after binding to the antigen. Since CD3ζ cannot provide a fully competent activation signal, additional co-stimulatory signaling may be required. Examples of co-stimulatory domains include endodomains from CD28, OX40, 4-1BB, CD27, and ICOS, which can be used with CD3ζ to transmit proliferation / survival signals.

[0124] In one embodiment, at least one co-stimulatory end-domain is used with CD3ζ. In a particular embodiment, the co-stimulatory end-domain is selected from the group consisting of end-domains from CD28, OX40, 4-1BB, CD27, and ICOS.

[0125] In another embodiment, at least two co-stimulatory end domains are used with CD3ζ. In a particular embodiment, the two co-stimulatory end domains are selected from the group consisting of end domains from CD28, OX40, 4-1BB, CD27, and ICOS in any combination and order. Particularly preferred combinations include end domains from CD28 and CD3ζ, end domains from OX40 and CD3ζ, end domains from 4-1BB and CD3ζ, end domains from CD28, OX40, and CD3ζ, and end domains from CD28, 4-1BB, and CD3ζ.

[0126] The transmembrane and intracellular T cell signaling domains (endodomains) of CARs having an activated endodomain may include the sequences shown in SEQ ID NOs. 13-18 or variants thereof having at least 80% sequence identity.

[0127] [ka]

[0128] [ka]

[0129] [ka] [ka]

[0130] [ka]

[0131] [ka]

[0132] [ka]

[0133] A variant sequence can have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with sequence numbers 13-18 if the sequence provides an effective transmembrane domain and an effective intracellular T cell signaling domain.

[0134] The CAR of the present invention may include sequences from the group of sequences shown in SEQ ID NOs. 25 to 36.

[0135] [ka]

[0136] [ka]

[0137] [ka]

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[0138]

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[0139]

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[0140]

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[0141]

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[0142]

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[0143]

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[0144]

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[0145]

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[0146]

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[0147] 4. Bispecific T cell Engagers Based on the fundamental concept of having two antibody-like binding domains, a wide variety of molecules have been developed.

[0148] Bispecific T-cell engagement molecules are a class of bispecific antibody-type molecules developed primarily for use as anticancer drugs. They direct the cytotoxic activity of the host immune system, more specifically T cells, towards target cells such as cancer cells. In these molecules, one binding domain binds to T cells via the CD3 receptor, while the other binds to target cells such as tumor cells (through tumor-specific molecules). Because bispecific molecules bind to both target cells and T cells, they bring target cells into close proximity with T cells, allowing T cells to exert their effects, such as cytotoxic effects on cancer cells. The formation of a T-cell:bispecific antibody:cancer cell complex induces signal transduction in T cells, leading to the release of cytotoxic mediators, for example. Ideally, the drug only needs to induce the desired signal transduction in the presence of target cells, leading to selective killing.

[0149] Bispecific T cell engagement molecules have been developed in several different formats, but one of the most common is a fusion consisting of two tandem-arranged single-chain variable fragments (scFv) of different antibodies. These are sometimes known as BiTEs (Bispecific T cell engagers).

[0150] The present invention also aims to provide a bispecific molecule capable of selectively recognizing TRBC2 and activating T cells. For example, the molecule may be BiTE.

[0151] Therefore, in another embodiment, the present invention provides a bispecific T cell engager (BiTE) comprising the variant antigen-binding domain and the T cell activation domain of the present invention, hereafter referred to as "the BiTE of the present invention".

[0152] The term “variant antigen-binding domain of the present invention” is described in detail in relation to the first aspect of the present invention, and its features and embodiments are equally applicable to this aspect of the present invention.

[0153] As used herein, the term "T cell activation domain" refers to a second domain capable of activating T cells. The T cell activation domain may be an scFv that specifically binds to CD3. Examples of anti-CD3 scFvs suitable for the present invention are well known in the art and include, without limitation, scFvs derived from OKT3.

[0154] Bispecific molecules can contain signal peptides that aid in their production. These signal peptides can induce the secretion of bispecific molecules by host cells, allowing the bispecific molecules to be collected from the supernatant of host cells.

[0155] The signal peptide can be located at the amino terminus of the molecule. A bispecific molecule can have the following general formula: signal peptide - variant antigen-binding domain of the present invention - T cell activation domain.

[0156] To link the variant antigen-binding domain and the T cell activation domain of the present invention and to spatially separate the two domains, the bispecific molecule may include a spacer sequence.

[0157] The spacer sequence may include, for example, an IgG1 hinge or a CD8 stalk. The linker may instead include an alternative linker sequence having similar length and / or domain spacing characteristics to the IgG1 hinge or CD8 stalk.

[0158] 5. Nucleic acids In another embodiment, the present invention also provides a nucleic acid sequence encoding a variant antigen-binding domain of the present invention, hereafter referred to as "the first nucleic acid of the present invention."

[0159] In another embodiment, the present invention also provides a nucleic acid sequence encoding the antibody of the present invention, hereafter referred to as "the second nucleic acid of the present invention."

[0160] In another embodiment, the present invention also provides a nucleic acid sequence encoding the CAR of the present invention, hereafter referred to as "the third nucleic acid of the present invention."

[0161] In another embodiment, the present invention also provides a nucleic acid sequence encoding the BiTE of the present invention, hereafter referred to as "the fourth nucleic acid of the present invention."

[0162] The terms “Variant antigen-binding domain of the present invention,” “Antibody of the present invention,” and “BiTE of the present invention” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments are equally applicable to those aspects of the present invention.

[0163] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” shall be synonymous with each other.

[0164] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, numerous different polynucleotides and nucleic acids may encode the same polypeptide. Furthermore, those skilled in the art should understand that, using conventional techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be made to reflect the codon usage of any particular host organism expressing the polypeptide.

[0165] The nucleic acid sequences and constructs of the present invention may contain alternative codons in regions of sequences encoding the same or similar amino acid sequences in order to avoid homologous recombination.

[0166] The nucleic acids according to the present invention may include DNA or RNA. The nucleic acids may be single-stranded or double-stranded. They may be polynucleotides containing synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate skeletons, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. It should be understood that polynucleotides can be modified by any method available in the art for use as described herein. Such modifications can be made to enhance the in vivo activity or lifetime of the polynucleotide of interest.

[0167] The terms “variant,” “homologous,” or “derivative” in relation to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acids to or from the sequence.

[0168] 6. Vector The present invention also provides vectors or vector kits comprising one or more nucleic acid sequences of the present invention. Such vectors can be used to introduce nucleic acid sequences into host cells so that they express the variant antigen-binding molecules, antibodies, CARs, or BiTEs of the present invention.

[0169] The terms “Variant antigen-binding domain of the present invention,” “Antibody of the present invention,” and “BiTE of the present invention” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments are equally applicable to those aspects of the present invention.

[0170] The vector may be, for example, a plasmid or viral vector, such as a retroviral vector or lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0171] The vector may be capable of transfecting or transducing cytolytic immune cells, such as T cells or NK cells.

[0172] 7.Cells Another aspect of the present invention relates to cells containing the CAR of the present invention, hereafter referred to as "cells of the present invention".

[0173] The cells may contain the nucleic acid or vector of the present invention.

[0174] The terms “CAR of the present invention,” “nucleic acid of the present invention,” and “vector of the present invention” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments are equally applicable to those aspects of the present invention.

[0175] The cells may be cytolytic immune cells, such as T cells or NK cells.

[0176] T cells, or T lymphocytes, are a type of lymphocyte that plays 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 T cell receptors (TCRs) on their cell surface. There are various types of T cells, as summarized below.

[0177] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when peptide antigens are presented 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 that promote different types of immune responses.

[0178] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted from regulatory T cells, CD8+ cells can be inactivated and put into an anerergic state, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0179] Memory T cells are a subset of antigen-specific T cells that persist for a long period after an infection has subsided. Upon re-exposure to a related antigen, they rapidly increase in number to become numerous effector T cells, thereby contributing to an immune system that "memorizes" past infections. Memory T cells comprise 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 either CD4+ or CD8+. Memory T cells generally express the cell surface protein CD45RO.

[0180] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary roles are to shut down T cell-mediated immunity and bring the immune response to an end, and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0181] Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells.

[0182] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are associated with interactions between developing T cells and bone marrow dendritic cells (CD11c+) and plasmacytoid dendritic cells (CD123+) activated by 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, which can lead to the fatal autoimmune disease IPEX.

[0183] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0184] The cell may be a natural killer cell (or NK cell). 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.

[0185] NK cells (which belong to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute a third type of cell that differentiates from a common lymphoid progenitor cell that gives rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus and then enter the circulation from there.

[0186] The cells of the present invention may be any of the cell types pointed out above. In one embodiment, the cells of the present invention are T cells. In another embodiment, the cells of the present invention are NK cells.

[0187] Cells according to this aspect of the present invention can be produced ex vivo from the patient's own peripheral blood (first party), or in the context of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0188] Alternatively, the cells according to this aspect of the invention may be derived from the ex vivo differentiation of inducible precursor cells or embryonic precursor cells into lytic cells. Alternatively, immortalized lytic cell lines that retain their lytic function and can act as therapeutic agents, such as T or NK cells, can be used.

[0189] In all these embodiments, the CAR-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of many means including transduction with viral vectors, transfection of DNA or RNA.

[0190] The cells of the invention may be ex vivo cells from a subject. The cells may 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, for example, by treatment with anti-CD3 monoclonal antibody, prior to transduction with the nucleic acid encoding the molecule providing the CAR of the invention.

[0191] The cells of the invention can be produced by a method comprising the step of transducing or transfecting the cells with a vector of the invention comprising a nucleic acid sequence encoding a CAR.

[0192] The method for producing the cells of the invention can further comprise the step of isolating the cells from a cell-containing sample from a subject or the other donor sources described above, prior to the transduction or transfection step. When the cells are lytic cells, the sample is a lytic cell-containing sample from a subject.

[0193] The term "subject" or "individual" used in connection with the present invention refers to a member of a mammalian species, preferably a male or female human of any age or race.

[0194] The cells of the invention can then be purified, for example, by selection based on the expression of the antigen-binding domain of the CAR.

[0195] 8. Conjugate The variant antigen-binding domain or antibody of the present invention may be a conjugate of the variant antigen-binding domain or antibody, for example, the conjugate may be a detectable entity or a chemotherapeutic entity.

[0196] The terms “Variant antigen-binding domain of the present invention” and “Antibody of the present invention” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments are equally applicable to those aspects of the present invention.

[0197] The detectable entity may be a fluorescent moiety, such as a fluorescent peptide. As used herein, the term "fluorescent peptide" refers to a polypeptide that emits light of a detectable wavelength after 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.

[0198] The variant antigen-binding domain or antibody of the present invention, conjugated with a detectable entity, can be used to determine the TRBCs of malignant T cells.

[0199] As used herein, the term "chemotherapeutic entity" refers to an entity that is destructive to cells, i.e., an entity that reduces the viability of cells. The resulting conjugate is hereafter referred to as "chemotherapeutic conjugate of the present invention." The chemotherapeutic entity may be a cytotoxic drug. The intended chemotherapeutic agents include alkylating agents, nitrosoureas, ethyleneimine / methylmelamine, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophyllotoxin, enzymes such as L-asparaginase; biological reaction modifiers, e.g., IFNα, IL-2, G-CSF and GM-CSF; platinum coordination complexes, e.g., cisplatin and carboplatin, anthracendione, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; corticosteroids, e.g., mitotane (o,p'-DDD) and aminoglutethimide; corticosteroid antagonists, e.g., pre Hormone and antagonists including donisone and its equivalents, dexamethasone and aminoglutethimide; progestins, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, e.g., diethylstilbestrol and ethinylestradiol equivalents; antiestrogenic drugs, e.g., tamoxifen; androgens, e.g., testosterone propionate and fluoxymesterone / equivalents; antiandrogenic drugs, e.g., flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogenic drugs, e.g., flutamide, without limitation.

[0200] The variant antigen-binding domain or antibody of the present invention, conjugated with a chemotherapeutic entity, enables targeted delivery of the chemotherapeutic entity to cells expressing TRBC2.

[0201] 9. Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition containing the cells or a plurality of cells of the present invention, or an antibody, or BiTE, or a chemotherapy conjugate, and hereafter referred to as "the pharmaceutical composition of the present invention."

[0202] The pharmaceutical composition may further contain pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition may optionally contain one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for, for example, intravenous infusion.

[0203] The terms “cells of the present invention,” “antibodies of the present invention,” “BiTE of the present invention,” and “chemotherapy conjugates of the present invention” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments are equally applicable to these aspects of the present invention.

[0204] Administration The administration of cells or a plurality of cells, or antibodies, or BiTE, or chemotherapy conjugates of the present invention can be achieved using any of the various routes that make the active ingredient bioavailable. For example, the drug can be administered via oral and parenteral routes, or through local delivery such as intraperitoneal, intravenous, subcutaneous, percutaneous, or intramuscular routes, for example, by catheter or stent.

[0205] Generally, a physician determines the most appropriate actual dosage for each individual patient, which will vary depending on the patient's age, weight, and response. The dosage should be sufficient to reduce or decrease the number of clonal T cells expressing TRBC1 or TRBC2.

[0206] 10. Treatment Method In another embodiment, the present invention provides cells, antibodies, or BiTEs, or conjugates for use in pharmaceuticals.

[0207] In another aspect, the present invention provides a method for treating T-cell lymphoma or leukemia in a subject, hereafter referred to as the "treatment method of the present invention," comprising the step of administering cells, antibodies, BiTE, or chemotherapeutic conjugates of the present invention to a subject, wherein malignant T cells express TRBC2. The administration step may be in the form of a pharmaceutical composition as described above.

[0208] This aspect of the invention can be formulated instead as a cell, or an antibody, or a BiTE, or a conjugate for chemotherapy of the invention for use in the treatment of T cell lymphoma or leukemia, hereinafter referred to as "the cell, antibody, BiTE or conjugate for chemotherapy of the invention", wherein the malignant T cells express TRBC2.

[0209] This aspect of the invention can be formulated instead as the use of a cell, or an antibody, or a BiTE, or a conjugate for chemotherapy of the invention in the manufacture of a medicament for treating T cell lymphoma or leukemia, wherein the malignant T cells express TRBC2.

[0210] The terms "cell of the invention", "antibody of the invention", "BiTE of the invention", "subject" and "conjugate for chemotherapy of the invention" are described in detail in the context of previous aspects of the invention, and their features and embodiments apply equally to these aspects of the invention.

[0211] A method for treating T cell lymphoma and / or leukemia relates to the therapeutic use of a cell, antibody, BiTE or conjugate for chemotherapy of the invention. As used herein, the cell, antibody, BiTE or conjugate for chemotherapy of the invention can be administered to a subject having existing T cell lymphoma and / or leukemia to alleviate, reduce or improve at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0212] Methods for preventing T-cell lymphoma and / or leukemia relate to the prophylactic use of cells, antibodies, BiTEs, or chemotherapy conjugates of the present invention. In this specification, such cells, antibodies, BiTEs, or chemotherapy conjugates may be administered to subjects who have not yet contracted T-cell lymphoma and / or leukemia, and / or who do not exhibit any symptoms of T-cell lymphoma and / or leukemia, in order to block or reduce the cause of the disease, or to reduce or prevent the occurrence of at least one symptom associated with the disease. Subjects may be predisposed to or at risk of developing T-cell lymphoma and / or leukemia.

[0213] The method may include the following steps: (i) A step of isolating a sample containing cytotoxic cells; (ii) the step of transfecting or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; and (iii) A step of administering cells from (ii) to the target cells.

[0214] Cytotoxic cell-containing samples can be isolated from the subject or from other sources, such as those described above. Cytotoxic cells such as T or NK cells can be isolated from the subject's own peripheral blood (first source), from the donor's peripheral blood (second source) in the context of hematopoietic stem cell transplantation, or from the peripheral blood of an unrelated donor (third source).

[0215] Methods for treating T-cell lymphoma and / or leukemia relate to the therapeutic use of drugs. In this specification, drugs may be administered to subjects with a pre-existing condition of T-cell lymphoma and / or leukemia to alleviate, reduce or improve at least one symptom associated with the disease, and / or to slow, reduce or block the progression of the disease.

[0216] These therapeutic applications include the administration of therapeutically effective doses of the cells, antibodies, BiTE, or chemotherapy conjugates of the present invention.

[0217] As used herein, the term “therapeutic dose” refers to the amount of the cells, antibodies, BiTE, or chemotherapy conjugates of the present invention required to achieve evaluable prevention, cure, delay, reduction of the severity of, or improvement of one or more symptoms of TRBC2-positive T-cell lymphoma and / or leukemia.

[0218] The method of the present invention can be used for the treatment of any lymphoma and / or leukemia associated with clonal proliferation of cells expressing a T cell receptor (TCR) containing TRBC2. Thus, the present invention relates to a method for treating diseases involving malignant T cells expressing a TCR containing TRBC2.

[0219] The method of the present invention can be used to treat T-cell lymphoma in which malignant T cells express a TCR containing TRBC2. "Lymphoma" is used herein by its standard meaning and refers to cancer that commonly occurs in the lymph nodes but can also affect the spleen, bone marrow, blood and other organs. Lymphoma commonly presents as a solid tumor of lymphocytes. The primary symptom associated with lymphoma is lymphadenopathy, while secondary (B) symptoms may include fever, night sweats, weight loss, loss of appetite, fatigue, shortness of breath and itching.

[0220] The method of the present invention can be used to treat T-cell leukemia in which malignant T cells express a TCR containing TRBC2. "Leukemia" is used herein in its standard sense and refers to cancer of the blood or bone marrow.

[0221] The following is an illustrative, non-exclusive list of diseases that can be treated by the method of the present invention.

[0222] Peripheral T-cell lymphoma Peripheral T-cell lymphomas are relatively rare lymphomas, accounting for less than 10% of all non-Hodgkin lymphomas (NHLs). However, they have an invasive clinical course, and the causes and precise cellular origins of the majority of T-cell lymphomas are still not fully defined.

[0223] Lymphoma typically first presents as swelling in the neck, armpits, or groin. Additional swelling may occur in other locations where lymph nodes are located, such as within the spleen. Generally, enlarged lymph nodes can encroach on blood vessels, nerves, or gastric space, potentially causing swelling, tingling, and numbness in the arms and legs, or a feeling of fullness, respectively. Nonspecific symptoms of lymphoma may also include fever, chills, unexplained weight loss, night sweats, lethargy, and itching.

[0224] The WHO classification uses morphological and immunophenotypic features, along with clinical characteristics and, in some cases, genetics, to provide a detailed categorization of peripheral T-cell lymphomas that is meaningful for prognosis and treatment (Swerdlow et al.; WHO classification of tumors of haematopoietic and lymphoid tissues. 4th edition; Lyon: IARC Press; 2008). The anatomical localization of neoplastic T cells is, in part, parallel to their proposed normal cell counterparts and functions, and therefore T-cell lymphomas are associated with lymph nodes and peripheral blood. This approach allows for a better understanding of some aspects of the manifestation of T-cell lymphoma, including cell distribution, some morphological characteristics, and associated clinical findings.

[0225] The most common type of T-cell lymphoma is peripheral T-cell lymphoma (PTCL-NOS), which accounts for 25% of all cases, followed by angioimmunoblastic T-cell lymphoma (AITL) at 18.5%.

[0226] Other unspecified peripheral T-cell lymphoma (PTCL-NOS) PTCL-NOS accounts for over 25% of all peripheral T-cell lymphomas and NK / T-cell lymphomas, making it the most common subtype. It is determined by exclusion and does not correspond to any of the specific mature T-cell lymphoma entities listed in the current WHO 2008. Therefore, it is similar to diffuse large B-cell lymphoma (DLBCL-NOS) which is not otherwise identified.

[0227] The majority of patients are adults, with a median age of 60 years and a male-to-female ratio of 2:1. While the majority of cases originate from nodules, approximately 13% of patients develop extranodal symptoms, most commonly involving the skin and gastrointestinal tract.

[0228] The cytological spectrum is very broad, ranging from pleomorphic to monomorphic. Three morphologically defined variants have been described, including the lymphoid epithelioid (Rennert) variant, the T-zone variant, and the follicular variant. The lymphoid epithelioid variant of PTCL contains abundant background epithelioid cell histiocytes and is generally CD8-positive. It is associated with a better prognosis. The follicular variant of PTCL-NOS is beginning to emerge as a potentially distinct clinicopathological entity.

[0229] The majority of PTCL-NOS cases exhibit a mature T-cell phenotype, and the majority of cases are CD4-positive. Loss of variability in at least one pan-T-cell marker (CD3, CD2, CD5, or CD7) is observed in 75% of cases, with CD7 and CD5 being most often downregulated. CD30 and, rarely, CD15 may be expressed, with CD15 being a feature of adverse prognosis. CD56 expression, though rare, also has a negative prognostic impact. Additional adverse pathological prognostic factors include proliferation rates exceeding 25% based on KI-67 expression and the presence of more than 70% transformed cells. Immunoexpressive analysis of these lymphomas yields limited insights into their biology.

[0230] Angioimmunoblastic T-cell lymphoma (AITL) AITL is a systemic disease characterized by pleomorphic infiltration involving lymph nodes, prominent high endothelial venules (HEVs), and perivascular enlargement of follicular dendritic cell (FDC) reticular structures. AITL is considered a de novo T-cell lymphoma originating from follicular helper αβ T cells (TFHs), which are usually found in germinal centers.

[0231] 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 in middle-aged to elderly adults, with a median age of 65 years, and the incidence is nearly equal in men and women. Clinically, patients usually have a progressive disease with generalized lymphadenopathy, hepatosplenomegaly, and marked constitutional symptoms. Itchy skin rashes are commonly present. Often, polyclonal hypergammaglobulinemia associated with an autoimmune phenomenon is present.

[0232] Three distinct morphological patterns have been described for AITL. In the early lesion of AITL (Pattern I), a conserved architecture is usually shown with characteristic hyperplastic follicles. Neoplastic proliferation is localized to the periphery of the follicle. In Pattern II, the nodular architecture is partially lost, and slightly vestigial follicles are retained. The subcapsular sinus is preserved and even enlarged. The paracortex contains branched HEVs, and FDCs proliferate beyond B-cell follicles. Neoplastic cells are small to medium in size, with minimal cytological atypicality. They often have clear to smudged cytoplasm and may exhibit a distinct cell membrane. A polymorphic inflammatory background is usually evident.

[0233] AITL is a T-cell malignancy, but it is characterized by a significant increase in B cells and plasma cells, which is thought to reflect the neoplastic cells' function as TFH cells. Both EBV-positive and EBV-negative B cells are present. In some cases, atypical B cells may morphologically and immunophenotypically resemble Hodgkin / Reed-Sternberg-like cells, sometimes leading to diagnostic confusion. B cell proliferation in AITL can be widespread, and in some patients develop secondary EBV-positive diffuse large B-cell lymphoma (DLBCL) or, more rarely, EBV-negative B-cell tumors, often with plasmacytic differentiation.

[0234] Neoplastic CD4-positive T cells in AITL exhibit potent 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 HEVs, B cell activation, plasmacytic differentiation, and increased FDC reticular structure, all contributing to the morphological and clinical features of AITL. Strong PD-1 expression in perifollicular tumor cells is particularly useful in distinguishing AITL pattern I from reactive follicle and paracortical hyperplasia.

[0235] The follicular variant of PTCL-NOS is a distinct entity possessing the TFH phenotype. In contrast to AITL, it lacks the extrafollicular enlargement of prominent HEV or FDC reticular structures. Neoplastic cells may form intrafollicular aggregates mimicking B-cell follicular lymphoma, but may also have interfollicular growth patterns or be accompanied by mantle enlargement. Clinically, the follicular variant of PTCL-NOS is distinct from AITL because patients more frequently present with initial disease involving partial lymph node involvement and may lack the constitutional symptoms associated with AITL.

[0236] Anaplastic large cell lymphoma (ALCL) ALCL can be subdivided into ALCL-"anaplastic lymphoma kinase" (ALK)+ or ALCL-ALK-.

[0237] ALCL-ALK+ is one of the most well-defined entities of peripheral T-cell lymphoma, possessing characteristic "special 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 the most common in the first 30 years of life. Patients often present with lymphadenopathy, but involvement in extranodal sites (skin, bone, soft tissue, lung, liver) and B symptoms are common.

[0238] ALCL and ALK+ exhibit a broad morphological spectrum, with five distinct patterns described, although all variants contain some characteristic cells. These characteristic cells have eccentric horseshoe-shaped or kidney-shaped nuclei and prominent perinuclear eosinophilic Golgi regions. Tumor cells grow in an adhesive pattern favoring sinus involvement. Smaller tumor cells are dominant in the small cell variant, while in the lymphohistiocytic variant, the presence of tumor cells is masked by abundant histiocytes, many of which are small.

[0239] By definition, all cases show ALK and CD30 positivity, with expression typically weaker in smaller tumor cells. Pan-T cell markers are often absent, and CD3 surface expression is absent in 75% of cases.

[0240] ALK expression is a characteristic recurrent genetic alteration resulting from the rearrangement of the ALK gene on chromosome 2 p23 to one of many partner genes, leading to the expression of a chimeric protein. The most common partner gene, occurring in 75% of cases, is nucleophosmin (NPM1) on chromosome 5 q35, resulting in t(2;5)(p23;q35). The cellular distribution of ALK in different translocation variants can vary depending on the partner gene.

[0241] ALCL-ALK- is included as a provisional category in the 2008 WHO classification. It is defined as a CD30-positive T-cell lymphoma that is morphologically indistinguishable from ALCL-ALK+, with an adhesive growth pattern and the presence of characteristic cells, but lacks ALK protein expression.

[0242] Patients are typically adults between 40 and 65 years of age, in contrast to ALCL-ALK+, which is more common in children and young adults. In ALCL-ALK-, both lymph nodes and extranodal tissues can be involved, although the latter is less common than in ALCL-ALK+. In the majority of ALCL-ALK- cases, the disappearance of the lymph node architecture by sheets of sticky neoplasm cells with typical "characteristic" features is demonstrated. In contrast to ALCL-ALK+, small cell morphological variants are not recognized.

[0243] In ALCL-ALK-, unlike its ALK+ counterpart, greater conservation of surface T cell marker expression is observed, but expression of cytotoxic markers and epithelial membrane antigens (EMAs) is less likely. Gene expression signatures and recurrent chromosomal imbalances differ between ALCL-ALK- and ALCL-ALK+, confirming that these are distinct entities at the molecular and genetic levels.

[0244] 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 for ALCL-ALK- is reported to be 49%, which is not as good as the 5-year overall survival rate for ALCL-ALK+ (70%), but at the same time, it is significantly better than the 5-year overall survival rate for PTCL-NOS (32%).

[0245] Enteropathy-associated T-cell lymphoma (EATL) EATL is an invasive neoplasm thought to originate from intraepithelial T cells in the intestine. (2008) The WHO classification recognizes two distinct types of EATL based on morphology, immunohistochemicals, and genetics: Type I (representing the majority of EATL cases) and Type II (accounting for 10-20% of cases).

[0246] Type I EATL is usually accompanied by overt or clinically asymptomatic gluten-irritable bowel syndrome and is more commonly seen in patients of Nordic descent, which is due to the high prevalence of celiac disease in this population.

[0247] Most commonly, EATL lesions are found in the jejunum or ileum (90% of cases), and rarely in the duodenum, colon, stomach, or areas outside the gastrointestinal tract. Intestinal lesions are usually multifocal and involve mucosal ulceration. The clinical course of EATL is invasive, and the vast majority of patients die within one year from the disease or its complications.

[0248] The cytological spectrum of EATL type I is broad, and some cases may contain undifferentiated cells. In some cases, there is a polymorphic inflammatory background that can obscure the neoplastic component. The intestinal mucosa in the area adjacent to the tumor often exhibits features of celiac disease, with villi smoothing and an increased number of intraepithelial lymphocytes (IELs), which may represent disease-causing progenitor cells.

[0249] Immunohistochemistry reveals that neoplastic cells are often 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. The mucosal homing receptor CD103 may be expressed in EATL.

[0250] Type II EATL, also known as monomorphic CD56+ intestinal T-cell lymphoma, is defined as a tumor of the intestine composed of small to medium-sized monomorphic T cells that express both CD8 and CD56. In most cases, the tumor spreads laterally within the mucosa, and there is no inflammatory background. The majority of cases express γδ TCRs, but some cases also exhibit αβ TCRs.

[0251] Type II EATL is more globally distributed than type I EATL and is often found in Asian or Latin American populations where celiac disease is rare. In individuals of European descent, EATL, type II accounts for approximately 20% of intestinal T-cell lymphomas and is associated with a history of celiac disease in at least a subset of cases. The clinical course is invasive.

[0252] Hepatosplenic T-cell lymphoma (HSTL) HSTLs are generally aggressive, systemic neoplasms originating from gammadelta-cytotoxic T cells of the innate immune system, although in rare cases they may originate from alpha-beta T cells. It is one of the rarest T-cell lymphomas, generally affecting adolescents and young adults (median age 35 years), and is strongly male-dominant.

[0253] Extranodal NK / T cell lymphoma nasal type Extranodal NK / T-cell lymphoma, nasal type, is an invasive disease, often accompanied by destructive midline lesions and necrosis. The majority of cases originate from NK cells, but some cases originate from cytotoxic T cells. It is universally associated with Epstein-Barr virus (EBV).

[0254] Cutaneous T-cell lymphoma The method of the present invention can also be used to treat cutaneous T-cell lymphoma.

[0255] Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells into the skin, resulting in various lesions. These lesions change shape as the disease progresses, typically beginning as a rash, eventually forming plaques and tumors before metastasizing to other parts of the body.

[0256] Examples of cutaneous T-cell lymphomas include those listed in the following exemplary, non-exclusive list: mycosis fungoides, Paget's reticular plaque, Sézary syndrome, granulomatous lasciform skin, lymphomatoid papulosis, chronic lichenoid pityriasis, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30- cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Rennard lymphoma, subcutaneous T-cell lymphoma, and vascular central lymphoma.

[0257] The signs and symptoms of CTCL vary depending on the specific disease, of which the two most common types are mycosis fungoides and Sézary syndrome. Classical mycosis fungoides has three stages: - Macular (atrophic or non-atrophic): Nonspecific dermatitis, macules on the lower trunk and buttocks; minimal or absent itching; - Plaque: Severely pruritic plaque, lymphadenopathy; and - Tumor: Prone to ulcer formation It can be divided into two categories.

[0258] Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymphadenopathy, hyperkeratosis of the palms and / or soles, alopecia, onychomycosis, ectropion, and hepatosplenomegaly.

[0259] Of all primary cutaneous lymphomas, 65% are T-cell lymphomas. The most common immunophenotype is CD4 positivity. The term cutaneous T-cell lymphoma encompasses a wide variety of disorders, so there is no common pathophysiology among these diseases.

[0260] The primary etiological mechanisms of cutaneous T-cell lymphoma (i.e., mycosis fungoides) are not yet understood. Mycosis fungoides may be preceded by a T-cell mediated chronic inflammatory skin disease, which in some cases can progress to a lethal lymphoma.

[0261] Primary cutaneous ALCL (C-ALCL) C-ALCL is often morphologically indistinguishable from ALC-ALK-. It is defined as a large cell cutaneous tumor in which more than 75% of cells express CD30 in an undifferentiated, pleomorphic, or immunoblastic form. Along with lymphomatoid papulosis (LyP), C-ALCL 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.

[0262] The immunohistochemical staining profile is quite similar to ALCL-ALK-, with a higher proportion of cases showing positivity for cytotoxic markers. At least 75% of tumor cells should be positive for CD30. CD15 is also expressed, and if lymph node involvement occurs, differentiation from classical Hodgkin lymphoma can be difficult. Rare cases of ALCL-ALK+ may present with localized cutaneous lesions and may resemble C-ALCL.

[0263] T-cell acute lymphoblastic leukemia T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% of ALL cases in the pediatric cohort and approximately 25% of ALL cases in the adult cohort. Patients typically have high white blood cell counts and may show organ enlargement, particularly mediastinal hypertrophy and involvement of the central nervous system (CNS).

[0264] The method of the present invention can be used to treat T-ALL associated with malignant T cells expressing TCRs containing TRBCs.

[0265] T-cell prolymphocytic leukemia T-cell prelymphocytic leukemia (T-PLL) is a mature T-cell leukemia characterized by invasive behavior and a preference for involvement of the blood, bone marrow, lymph nodes, liver, spleen, and skin. T-PLL primarily affects adults over 30 years of age. Other names include chronic T-cell lymphocytic leukemia, nodular T-cell leukemia, and T-cell prelymphocytic leukemia / T-cell lymphocytic leukemia.

[0266] In peripheral blood, T-PLL consists of medium-sized lymphocytes with a single nucleolus and basophilic cytoplasm, sometimes accompanied by blebs or projections. The nuclei are typically round to oval in shape, and patients may have cells with more irregular nuclear contours, similar to the cerebral-like nuclear shape seen in Sézary syndrome. Small cell variants account for 20% of all T-PLL cases, while Sézary cell-like (cerebral-like) variants are found in 5% of cases.

[0267] T-PLL has an immunophenotype of mature (postthymic) T lymphocytes, and neoplastic cells are generally positive for pan-T antigens CD2, CD3, and CD7, and negative for TdT and CD1a. The CD4+ / CD8- immunophenotype is present in 60% of cases, the CD4+ / CD8+ immunophenotype in 25%, and the CD4- / CD8+ immunophenotype in 15% of cases.

[0268] T-cell lymphomas or leukemias that should be treated or prevented may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0269] The treatment method may include the step of administering a therapeutic dose of the cells or a plurality of cells of the present invention, or an antibody, or BiTE, or a chemotherapy conjugate. Those skilled in the art can determine the amount of the cells or a plurality of cells of the present invention, or an antibody, or BiTE, or a chemotherapy conjugate that can exert a therapeutic effect on a patient by conventional methods.

[0270] 11. Diagnostic agents TRBC2 + TRBC1 +The proportion of T cells from healthy donors was previously determined to be 35% versus 65%, i.e., the central percentage of all T cells expressing TRBC1 was 35% (range, 25-47%) (Maciocia et al., 2017, Nat Med 23:1416-23). T cell lymphoma or leukemia is a clonal cancer (Maciocia et al., 2017; supra), so the unrestricted proliferation of malignant T cells, which is characteristic of T cell lymphoma or leukemia, results in a significantly distorted proportion of TRBC2 + or TRBC1 + T cells (i.e., TRBC2 - or TRBC1 - T cells). Thus, by specifically binding to TRBC2 and thus being able to distinguish between TRBC1 and TRBC2, the variant antigen-binding domains and antibodies of the present invention are useful agents for the diagnosis of T cell lymphoma or leukemia.

[0271] Thus, in another aspect, the present invention provides a diagnostic agent comprising the variant antigen-binding domain of the present invention, hereinafter referred to as "the first diagnostic agent of the present invention".

[0272] In another aspect, the present invention provides a diagnostic agent comprising the antibody of the present invention, hereinafter referred to as "the second diagnostic agent of the present invention". The terms "variant antigen-binding domain of the present invention" and "antibody of the present invention" are 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.

[0273] The variant antigen-binding domains or antibodies of the present invention used in these assays may be labeled or unlabeled. As used herein, the terms “detectable label” or “labeling agent” refer to molecular labeling that enables detection, localization, and / or identification of the molecule to which it is attached, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means, using suitable procedures and apparatus for detection. Suitable labeling agents for labeling antibodies include radionuclides, enzymes, phosphors, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and derivatives. As those skilled in the art will understand, unlabeled variant antigen-binding domains and antibodies need to be detected with additional reagents, such as labeled secondary antibodies. This is particularly beneficial for increasing the sensitivity of the detection method because it allows for signal amplification. There are a variety of conventional assays that can be used in the present invention, using 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), immunocytochemical and immunohistochemical techniques, flow cytometry, or multiplex detection techniques based on the use of protein microspheres, biochips or microarrays containing antibodies of the present invention. Other methods for detecting and quantifying TRBC2 using the variant antigen-binding domain or antibody of the present invention include affinity chromatography techniques or ligand-binding assays.

[0274] The diagnostic agent can be used to diagnose T-cell lymphoma or leukemia.

[0275] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0276] 12. Diagnostic Methods In another aspect, the present invention provides a method for diagnosing T-cell lymphoma or leukemia in a subject, hereafter referred to as the "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.

[0277] The terms “Variant antigen-binding domain of the present invention,” “Antibody of the present invention,” and “Subject” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0278] The diagnostic method of the present invention may further include the step of determining the percentage of TRBC2-positive T cells in a sample.

[0279] To carry out the first method of the present invention, a sample containing T cells, such as a biological sample, is obtained from the subject of study. As used herein, the terms “sample” or “biological sample” include different types of biological fluids or tissue sections of an affected organ containing T cells. Exemplary, non-limiting examples of samples useful in the diagnostic method of the present invention include different types of biological fluids containing T cells, such as blood, lymph, and cerebrospinal fluid. These biological fluid samples are obtained by any conventional method known to those skilled in the art. Alternatively, the sample may be a section of an affected organ tissue sample obtained by any conventional method, such as by biopsy or surgical excision, for example, from lymph nodes, spleen, tonsils, or thymus, and from frozen sections taken for histological purposes.

[0280] In the first step of the diagnostic method of the present invention, the variant antigen-binding domain or antibody of the present invention is brought into contact with a sample from a study subject under preferred conditions known to those skilled in the art.

[0281] Those skilled in the art can use several conventional methods suitable for performing the second step of the diagnostic method of the present invention to detect TRBC2 in a sample. Immunological methods are particularly beneficial. Therefore, the use of the first or second diagnostic agent of the present invention may be particularly beneficial for performing the diagnostic method according to the present invention. The features and specific embodiments of the diagnostic agent of the present invention have been previously defined and apply equally to the diagnostic method of the present invention.

[0282] In the diagnostic method of the present invention, TRBC2-positive T cells in a sample representing 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher percentages can indicate the presence of T-cell lymphoma or leukemia.

[0283] As will be understood by those skilled in the art, the prediction is preferably, but not required, correct in 100% of the subjects being diagnosed or evaluated. However, this term requires that it be possible to identify an increased probability that a statistically significant portion of the subjects will have a given outcome. Whether the data obtained from the subjects are statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, cross-validation classification evaluation, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. 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 lower.

[0284] Furthermore, given their ability to specifically bind to TRBC2-positive T cells, the variant antigen-binding domains or antibodies of the present invention can also be used in the 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 processes used to create images of the body (or its parts and functions), e.g., the human body, for clinical purposes, e.g., medical procedures that seek to reveal, diagnose, or examine a disease.

[0285] For this purpose, the variant antigen-binding domain or antibody of the present invention is labeled by a preferred method known in the art and provided as a drug for diagnostic imaging methods, such as radioimmunodiagnosis, positron emission tomography (PET), and endoscopic immunofluorescence, by conjugation and / or administration with a suitable molecule, such as a radioisotope or fluorescent dye. The variant antigen-binding domain and antibody of the present invention can be conjugated to a gamma radioisotope and used in radioimmunoscintigraphy using a gamma camera or single-photon tomography. The variant antigen-binding domain and antibody of the present invention can be conjugated to a positron emitter and used in PET. The variant antigen-binding domain and antibody of the present invention can be conjugated to a fluorescent dye such as Cy3, Cy2, Cy5, or FITC and used in endoscopic immunofluorescence. The variant antigen-binding domain and antibody of the present invention, modified as described, can be administered to an individual in an appropriate dose via any preferred route, such as intravenously, and the location of TRBC2-positive T cells can be detected, determined, or measured by a process known in the art. The methods and techniques used herein, including diagnostic imaging, are known to those skilled in the art, and they can also provide suitable dosage formulations.

[0286] The T-cell lymphoma or leukemia to be diagnosed may be selected from the following: peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0287] The sample may be a blood sample or derived from one.

[0288] 13. Personalized medication methods In another aspect, the present invention provides a method for identifying subjects of T-cell lymphoma or leukemia who are eligible for treatment with the cells, antibodies, BiTE or chemotherapy conjugates of the present invention, hereafter referred to as "the first method of the personalized drug of the present invention," the method comprising the step of determining the percentage of TRBC2-positive T cells in a sample containing T cells from the subject.

[0289] The terms “cells of the present invention,” “antibodies of the present invention,” “BiTE of the present invention,” “chemotherapeutic agents of the present invention,” “subjects,” and “samples containing T cells” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0290] In the first method of the personalized drug of the present invention, a subject is eligible for treatment with the cells, antibodies, BiTE or chemotherapy conjugate of the present invention if the percentage of TRBC2-positive T cells in the sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher.

[0291] In another aspect, the present invention provides a method for selecting a therapy comprising the cells, antibodies, BiTE or chemotherapy conjugate of the present invention for treatment of a subject, hereafter referred to as "the second method of personalized drug of the present invention," the method comprising the step of determining the percentage of TRBC2-positive T cells in a sample comprising T cells from a subject.

[0292] The terms “cells of the present invention,” “antibodies of the present invention,” “BiTE of the present invention,” “chemotherapeutic agents of the present invention,” “subjects,” and “samples containing T cells” are described in detail in relation to the prior aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0293] In a second method of personalized medicine according to the present invention, if the percentage of TRBC2-positive T cells in a sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher, a therapy comprising cells, antibodies, BiTE, or chemotherapy conjugates according to the present invention is selected to treat the subject.

[0294] The sample may be a blood sample or derived from one.

[0295] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified; 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 prelymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0296] The present invention will now be further described by examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended to limit the scope of the invention. [Examples]

[0297] (Example 1) Analysis of anti-TRBC2 antibody binding by surface plasmon resonance (SPR) The crystal structure of the TRBC1-specific monoclonal antibody hJovi-1 was elucidated to 2.4 Å in complex with the TRBC1-TCR (Figure 3). Through computational biology and protein engineering, several mutant versions of the anti-TRBC1 conjugate were rationally designed to switch specificity from TRBC1 to TRBC2. Several anti-TRBC2 conjugates were generated in IgG format. Table 1 shows details of the mutations contained in the VH and VL domains of hJovi-1.

[0298] Using a Biacore T200 instrument, Series S CM5 sensor tips were immobilized by amine bonding, and anti-human Fc capture antibody was applied to a density of 9000–10000 RU. HBS-P+ buffer was used as the liquid buffer under all experimental conditions. Test anti-TRBC2 antibody (Table 1) was captured on flow cells 2, 3, and 4 to a density of 100–300 RU. Recombinant purified TRBC1 or TRBC2 of known concentrations was used as the "analyte" and injected onto each flow cell at a flow rate of 30 μl / min with a contact time of 150 seconds and dissociation time of 300 seconds. In each experiment, flow cell 1 was unmodified and used for reference subtraction. To account for drift, a "zero concentration" sensorgram with only buffer was used for double reference subtraction. The data were fitted to a 1:1 Langmuir-coupled model. Because a capture system was used, local R was applied for data fitting in each case. max Parameters were used.

[0299] Table 1 shows the affinity results for TRBC1 and TRBC2 obtained for each anti-TRBC2 antibody. The test anti-TRBC2 antibodies generally showed preferential binding to TRBC2 in the nM range, and their ka and kd kinetics varied. In most cases, binding to TRBC1 was determined to be >1 μM, and the values ​​were close to the sensitivity limit of the instrument.

[0300] (Example 2) Generation of anti-TRBC2 CARs based on KFN binders Second-generation CAR constructs were generated based on the anti-TRBC2 triple mutant (T28K, Y32F, A100N mutation in the VH domain of hJovi-1, SEQ ID NO: 26) and humanized Jovi-1 (hJovi-1) (Figure 4). These CAR constructs were cloned into retroviral vectors and used to transduce activated PBMCs obtained from healthy donors.

[0301] (Example 3) Functional characterization of anti-TRBC2 CAR: cytokine generation To investigate the functional capacity 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 IFN-γ production was measured by ELISA. In Figure 5A, anti-TRBC2 triple mutant CAR-T cells showed greater IFN-γ release in the presence of TRBC2 ligand compared to TRBC1. In contrast, hJovi-1 CAR-T cells showed high IFN-γ production only when cultured with TRBC1 ligand (Figure 5B). These results demonstrate that CAR-T cells transduced with the anti-TRBC2 triple mutant have greater activation and cytokine release in response to TRBC2, but not when exposed to TRBC1.

[0302] (Example 4) Functional characterization of anti-TRBC2 CAR: Cytotoxic assay To determine the ability of anti-TRBC2 triple mutants to target TRBC2, a cytotoxic assay was set up using Raji cells transduced to express either 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 used to create Raji WT and Raji TRBC1 + Or Raji TRBC2 +The cells were cultured in a 1:1 (E:T) ratio with either of the other cells. Flow cytometry was used to measure the recovery of target cells 72 hours after culturing and to establish the cytotoxic capacity of the CAR-T cells. Cultures containing anti-TRBC2 triple mutant CAR-T cells were used with Raji TRBC2 + This showed limited survival of target cells (Figure 6). In contrast, anti-TRBC2 triple mutant CAR-T cells showed limited survival of Raji TRBC1 + Without eliminating target cells, TRBC2 + This demonstrates their increased ability to target cells.

[0303] We used engineered monoclonal antibodies to generate second-generation anti-TRBC2 CARs. We demonstrated that our anti-TRBC2 CARs exhibited specificity, cytokine release, and cytotoxicity against TRBC2+ cell lines in 72-hour co-culture, but not against TRBC1+ cell lines or cell lines that did not express a surface TCR. Anti-TRBC2 CAR T cells also demonstrated proliferative capacity in long-term co-culture assays.

[0304] (Example 5) Functional characterization of anti-TRBC2 CAR: lethal assay Based on anti-TRBC2 conjugates from Table 1 with the following mutations, we generated additional second-generation CAR constructs: - T28K, Y32F, and A100N mutations in the VH domain of hJovi-1 and N35K (referred to as N35K, SEQ ID NO: 25) in the VL domain, - T28K, Y32F, A100N, N103L mutations (referred to as N103L, SEQ ID NO: 27) in the VH domain of hJovi-1, - 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), - 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), - T28K, Y32F, A100N, Y102L, and 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).

[0305] These CAR constructs and the anti-TRBC2 triple mutant (T28K, Y32F, A100N mutation in the VH domain of hJovi-1, referred to as KFN, SEQ ID NO: 26) were cloned into retroviral vectors and used to transduce (a) Jurkat cells or (b) activated PBMCs obtained from healthy donors. The anti-TRBC1 control CAR, i.e., the hJovi-1 CAR construct, was used as a control in a killing assay using activated PBMCs.

[0306] a) Jurkat cells To evaluate whether the generated binder was specific to the TRBC2 antigen, the above anti-TRBC2 CAR construct was transduced into TRBC1+ Jurkat cells. Transduced HPB-ALL cells expressing only TRBC1 or TRBC2 (referred to as HPB TRBC1 and HPB TRBC2, respectively) were used as target cells, and transduced Jurkat cells were co-cultured in a 1:1 effector-to-target (E:T) ratio with HPB-ALL cells with knockout TCRs (referred to as HPB KO) as a negative control. Transduced Jurkat cells were plated alone or with αCD3 / αCD28 antibodies for use as negative or positive assay controls, respectively. Antigen-specific activation was investigated by flow cytometry after 24 hours, through CD69 staining.

[0307] The results shown in Figure 7 reveal that all transduced Jurkat cells were selectively activated after co-incubation with HPB TRBC2 cells. Since no CD69 upregulation was observed when Jurkat was co-cultured with TRBC1+ or HPB KO targets, these results demonstrate that all CARs tested exhibited specificity for TRBC2 targets.

[0308] b) PBMC Firstly, after staining with a 10 ug / ml biotinylated mouse JOVI-1 antibody that binds exclusively to TRBC1, PBMCs were separated into TRBC1+ and TRBC2+ cells using beads coated with magnetic anti-biotin. The TRBC2+ population was transduced with hJovi-1 CAR (JOVI), and the TRBC1+ population was transduced with the aforementioned anti-TRBC2 CAR. This differential transduction ensured that antigen activation and target killing were induced only when the target was added under controlled culture conditions (e.g., effector-to-target ratio and time point), rather than in mixed populations where conditions differed in each PBMC donor.

[0309] Secondly, HPB-ALL cells transduced to express only TRBC1 or TRBC2 (referred to as TRBC1+HPB-ALL and TRBC2+HPB-ALL, respectively) were used as target cells, and HPB-ALL cells with knockout TCRs (referred to as HPB-KO) were used as a negative control to set up a cell killing assay. Ten days after transduction, the cells were co-cultured in an E:T ratio of 1:2. Cell killing was investigated by flow cytometry 72 hours after assay setup.

[0310] The results demonstrated that hJovi-1 CAR-T cells exclusively killed TRBC1+ HPB-ALL cells, and all anti-TRBC2 CAR-T cells exclusively killed TRBC2+ HPB-ALL cells (Figure 8). Similar levels of background killing were observed for all TRBC1 and TRBC2-specific CAR-T cells in HPB-KO control cells, indicating that all CARs tested were specific to their respective cognitive antigens.

[0311] (Example 6) Analysis of the effects of different antibody formats on the binding of anti-TRBC2 antibodies by surface plasmon resonance (SPR). The effects of polymerization in anti-TRBC2 conjugates were analyzed by SPR. For this purpose, anti-TRBC2 antibodies with T28K, Y32F, A100N, Y102L, and N103M mutations in the VH domain and N35R mutations in the VL domain of the hJovi-1 antibody were used in three different formats: scFv (SEQ ID NO: 22), scFv-Fc (SEQ ID NO: 23), and scFv-COMP (SEQ ID NO: 24) antibody formats. [ka] [ka]

[0312] To test the 1:1 binding interaction of anti-TRBC2 scFv, a Biacore T200 instrument was used, with Series S CM5 sensor tips immobilized by amine bonding, and anti-human Fc capture antibody at a density of 9000–10000 RU. HBS-P+ buffer was used as the liquid buffer under all experimental conditions. The test anti-TRBC2 antibody was captured on flow 4 to a density of 200–250 RU. Recombinant purified TRBC1 or TRBC2 at known concentrations was used as the "analyte" and injected onto the respective flow cells at a flow rate of 30 μl / min with a contact time of 150 seconds and dissociation time of 300 seconds. Flow cell 1 was unmodified and used for reference subtraction. A "zero concentration" sensorgram with only buffer was used for double reference subtraction to account for drift. The data were fitted to a 1:1 Langmuir binding model. Because a capture system was used, local R was used for data fitting in each case. max Parameters were used. The dissociation rate value was determined, and the half-life was set to t. 1 / 2 It was calculated using =ln2 / kd.

[0313] The results shown in Figure 9A reveal that this anti-TRBC2 scFv binder has a half-life of 8 seconds. The binding affinity of this binder is shown in Table 1.

[0314] To test the multivalent interaction of scFv-Fc and scFv-COMP antibody formats, Series S CAP tips were immobilized with biotin capture reagent to 2500–5000 RU. Soluble recombinant biotinylated TRBC1 and TRBC2 were captured on flow cell 3 to densities of 90–110 RU in independent experimental cycles. Purified anti-TRBC2 scFv-Fc or scFv-COMP antibody was injected onto the flow cell at a flow rate of 30 μl / min with a contact time of 150 seconds and dissociation time of 300 seconds. In flow cell 1, biotinylated protein was omitted and used for reference subtraction. To account for drift, a buffer-only "0 concentration" sensorgram was used as a double reference subtraction. The data were fitted to a 1:1 Langmuir binding model. Since a capture system was used, the local Rmax parameter was used for data fitting in each case. Dissociation rate values ​​were determined, and the half-life was set to t 1 / 2 It was calculated using =ln2 / kd.

[0315] The results shown in Figures 9B and 9C reveal that the half-lives of the scFv-Fc and scFv-COMP antibody formats were 16.2 seconds and 7.3 minutes, respectively.

[0316] Therefore, these results demonstrate that polymerization improves the binding of low-affinity / high-specificity TRBC2 antibodies.

[0317] This application claims the rights of UK Patent Application No. 1817822.8, filed on 31 October 2018. This application is incorporated in its entirety by reference.

[0318] All publications described herein are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention is described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described methods for carrying out the invention, which will be apparent to those skilled in the art of molecular biology or related fields, are within the scope of the following claims.

Claims

[Claim 1] The invention described herein.

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