TRBCβ antibody conjugate

Abstract

The present disclosure provides a conjugate of an antibody that specifically binds to the TCR beta chain constant region (TRBC), the antibody having a fast dissociation rate constant (k d The present invention further provides medical uses and methods of personalized medicine utilizing the products of the present invention. The present invention provides, for example, antibody conjugates that specifically bind to the TCR beta chain constant region (TRBC), wherein the antibody has a specificity of 0.001 seconds. -1 ~0.3 seconds -1 The dissociation rate constant (k d )

Description

[Technical Field]

[0001] Field of Invention The present invention relates to an antibody conjugate in which an antibody specifically binds to the constant region (TRBC) of the TCRβ chain, wherein the antibody has a fast dissociation rate constant (kd). [Background technology]

[0002] Background of the Invention Lymphoid malignancies can be broadly divided into those originating from either T cells or B cells. T-cell malignancies are a clinically and biologically heterogeneous group of 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, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); and anaplastic large cell lymphoma (ALCL). Of all acute lymphoblastic leukemias (ALL), approximately 20% are T-cell phenotypes.

[0003] These symptoms typically behave more invasively compared to, for example, B-cell malignancies, with an estimated 5-year survival rate of only 30%. In T-cell lymphomas, a high proportion of patients present with disseminated disease, unfavorable International Prognostic Index (IPI) scores, and a high prevalence 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, where immunotherapies such as the anti-CD20 monoclonal antibody rituximab have dramatically improved outcomes, there are currently no equally effective, minimally toxic immunotherapies available for the treatment of T-cell malignancies. A significant challenge in the development of immunotherapies for T-cell damage is the considerable overlap in marker expression between clonal and normal T cells, and there is no single antigen that can clearly identify clonal (malignant) cells.

[0005] Targeting strategies based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2) have been reported (International Publication No. 2015 / 132598; Maciocia et al., 2017, Nat Med 23:1416-23). ​​Furthermore, it has been demonstrated that CARs targeting either TRBC1 or TRBC2 may provide an acceptable toxicity profile while conferring the ability to treat T cell lymphoma (Maciocia et al., 2017, Nat Med, 23:1416-23; International Publication No. 2015 / 132598).

[0006] However, diseases such as human T-cell leukemia virus, type 1 (HTLV-1)-associated leukemia and lymphoma may not be well-suited to cell therapy, despite the potential for treatment with TRBC1 / TRBC2-targeted drugs. This is because cell-based therapies may have a tendency towards T-cell-mediated internetic reactions.

[0007] Therefore, in this field, there is a need to provide alternative targeted agents that overcome the potential shortcomings of cell-based therapies in the treatment of T-cell lymphoma and leukemia.

[0008] Antibody-drug conjugates (ADCs) offer another form of immunotherapy that can be used to target T-cell lymphomas. ADCs offer further advantages over cell-based therapies, particularly in that they are less prone to T-cell-mediated friendly fire. Furthermore, ADCs may offer improved management of side effects.

[0009] The efficiency of ADC strategies largely depends on the internal translocation of cytotoxic conjugates into cancer cells. While ADCs based on TRBC-specific antibodies have been previously described (International Publication No. 2015 / 132598), their internal translocation characteristics remain unknown.

[0010] Important characteristics of antibodies suitable for ADCs include specific binding to TRBCs and high internal distribution capacity. The internal distribution capacity of an antibody depends on the properties of both the target antigen and the antibody. Predicting an antigen-binding site suitable for internal distribution from the molecular structure of the target, or predicting an antibody with high internal distribution capacity from its binding strength and physical properties, is difficult. Therefore, a crucial challenge in developing highly potent ADCs is obtaining antibodies with high internal distribution capacity to the target antigen.

[0011] Therefore, in this field, there is a need for a TRBC-specific ADC with optimal internal migration characteristics. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] International Publication No. 2015 / 132598 [Non-patent literature]

[0013] [Non-Patent Document 1] Maciocia et al., 2017, Nat Med 23:1416-23 [Overview of the project] [Means for solving the problem]

[0014] Overview of the Invention The inventors investigated ADC therapeutic strategies for T-cell lymphoma and leukemia using numerous TRBC-specific antibodies of varying affinities, with the aim of elucidating the internal migration characteristics of TRBC-specific antibodies upon binding to T cells. The results obtained helped identify binding affinities that confer good internal migration characteristics to specific antibodies. Unexpectedly, the inventors determined that a fast dissociation rate constant is important for achieving good internal migration of antibodies. These results contrast with the generally accepted view in the art, namely, that high-affinity antibodies, and therefore antibodies with slow dissociation rate constants, exhibit high internal migration ability.

[0015] Therefore, in the first aspect, the present invention relates to an antibody conjugate that specifically binds to the TCRβ chain constant region (TRBC), wherein the antibody is 0.001 seconds -1 ~0.3 seconds -1 Dissociation rate constant (k) in the range d The present invention provides an antibody conjugate having )

[0016] The antibody took 0.002 seconds. -1 ~0.1 seconds -1 k within the range d It may have.

[0017] Antibodies can be conjugated with chemotherapeutic entities, radionuclides, or detection entities.

[0018] The actual chemotherapy agent could be a tubulin inhibitor.

[0019] Tubulin inhibitors can be MMAEs.

[0020] Compared to the internal migration of 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, the antibody conjugate may exhibit increased internal migration upon binding to target cells.

[0021] Antibodies can specifically bind to TRBC1.

[0022] 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, the TRBC1-specific antibody exhibits the following mutations: - G106A in the aforementioned VH domain; -Y32F in the aforementioned VH domain; - G31S in the aforementioned VH domain; - G26P and T28K in the VH domain; -Y102M in the VH domain It may include one of the following.

[0023] 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, the TRBC1-specific antibody exhibits the following mutations: - G106A in the VH domain It may include.

[0024] Antibodies can specifically bind to TRBC2.

[0025] 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, the TRBC2-specific antibody exhibits the following mutation combinations: -T28K, Y32F, A100N, Y102L and N103M in the VH domain, and VL N35R in the VL domain; -T28K, Y32F, A100N in the VH domain; or -T28R, Y32F, A100N in the VH domain It may include one of the following.

[0026] 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, the TRBC2-specific antibody exhibits the following mutations: -T28K, Y32F, A100N, Y102L and N103M in the VH domain, and VL N35R in the VL domain It may include. In a second aspect, the present invention provides an antibody conjugate according to the first aspect of the present invention for use in the treatment of T-cell lymphoma or leukemia.

[0027] Treatment of T-cell lymphoma or leukemia in a subject may include administering an antibody conjugate to the subject to cause selective depletion of malignant T cells, along with normal T cells that express the same TRBCs as malignant T cells, but not to cause depletion of normal T cells that express TRBCs not expressed by malignant T cells.

[0028] The method may further include a step of examining the TCRβ chain constant region (TCRB) of malignant T cells derived from the target to determine whether the malignant T cells express TRBC1 or TRBC2.

[0029] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); anaplastic large cell lymphoma (ALCL); enteropathy-associated T-cell lymphoma (EATL); hepatosplenic T-cell lymphoma (HSTL); extranodal NK / T-cell lymphoma nasal type; cutaneous T-cell lymphoma; primary cutaneous ALCL; T-cell prolymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0030] In a third aspect, the present invention provides an antibody conjugate according to an aspect of the first aspect of the present invention for use in a method for targeting the delivery of a chemotherapeutic agent to cells expressing TRBC in a subject.

[0031] In a fourth aspect, the present invention provides a pharmaceutical composition comprising an antibody conjugate according to the first aspect of the present invention and a pharmaceutically acceptable carrier, diluent, excipient, or auxiliary agent.

[0032] In the fifth aspect, the present invention relates to a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, i) To determine whether malignant T cells in samples isolated from the subject express TRBC1 or TRBC2; ii) Selecting an antibody conjugate for use according to the second aspect of the present invention based on the TRBC1 or TRBC2 expression of malignant T cells; This provides a method that includes [something].

[0033] In the sixth aspect, the present invention provides a method for selecting a subject suffering from T-cell lymphoma or leukemia to receive a therapy comprising an antibody conjugate for use according to the second aspect of the present invention, i) To determine whether malignant T cells in samples isolated from the subject express TRBC1 or TRBC2; ii) Selecting subjects to receive antibody conjugate-based therapy for use according to the second aspect of the present invention, based on the TRBC1 or TRBC2 expression of malignant T cells; This provides a method that includes [something]. [Brief explanation of the drawing]

[0034] [Figure 1] Schematic diagram of 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. Four proteins of the CD3 complex (CD3ζ, CD3γ, CD3ε, and CD3δ) surround the T cell receptor (TCR). This TCR confers 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]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), diverse (D), ligated (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. T cells express many copies of a single TCR on their surface; therefore, each T cell expresses a TCR in which its β-chain constant region is encoded by either TRBC1 or TRBC2. [Figure 3] Structural interface between TCRβ and the Fab fragment of the TRBC1-specific antibody Jovi-1. [Figure 4] Internal migration profiles of pHrodo red-conjugated Jovi-1, KFN, and anti-HEL antibodies in HPB-ALL TRBC1, HPB-ALL TRBC2, and HPB-ALL KO cells. [Figure 5-1] Surface plasmon resonance experiments were performed on various mutated antibody variants to confirm affinity, association, and dissociation rates. [Figure 5-2] Surface plasmon resonance experiments were performed on various mutated antibody variants to confirm affinity, association, and dissociation rates. [Figure 5-3] Surface plasmon resonance experiments were performed on various mutated antibody variants to confirm affinity, association, and dissociation rates. [Figure 5-4] Surface plasmon resonance experiments were performed on various mutated antibody variants to confirm affinity, association, and dissociation rates. [Figure 6] Analysis of the binding kinetics of mutated antibody variants. Several binders were identified that have very similar association rates but different dissociation rates. [Figure 7]Internal migration profiles of pHrodo green-conjugated mutants of aTRBC1 in HPB-ALL TRBC1 and KO cells at 9 hours. Clones were selected while increasing the dissociation rate to TRBC1. Flow cytometry shows improved internal migration of the conjugate with faster dissociation rates up to Mut13 before internal migration is affected by a faster off-rate. Antibody mutants are listed in Tables 1 and 2. [Figure 8] Internal transfer of optimal affinity TRBC1 and TRBC2 antibodies in HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO cells. Antibody variants are listed in Tables 1 and 2. [Figure 9A] A. Schematic representation of anti-TCR PAB-MMAE conjugation via MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the antibody's ability to internalize. MFI: mean fluorescence intensity. C. Cytotoxic assays against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO with MMAE-conjugated Mut11, Mut15, and anti-HEL. ****P<0.0001 by fit comparison against HPB-ALL TCR KO. Antibody variants are listed in Tables 1 and 2. [Figure 9B] A. Schematic representation of anti-TCR PAB-MMAE conjugation via MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the antibody's ability to internalize. MFI: mean fluorescence intensity. C. Cytotoxic assays against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO with MMAE-conjugated Mut11, Mut15, and anti-HEL. ****P<0.0001 by fit comparison against HPB-ALL TCR KO. Antibody variants are listed in Tables 1 and 2. [Figure 9C]A. Schematic representation of anti-TCR PAB-MMAE conjugation via MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the antibody's ability to internalize. MFI: mean fluorescence intensity. C. Cytotoxic assays against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO with MMAE-conjugated Mut11, Mut15, and anti-HEL. ****P<0.0001 by fit comparison against HPB-ALL TCR KO. Antibody variants are listed in Tables 1 and 2. [Figure 10] Schematic diagram of the structure of TRBC1 and TRBC2-specific chimeric antigen receptors (CARs). [Modes for carrying out the invention]

[0035] Detailed description of the invention The present invention provides an antibody conjugate that specifically binds to the TCRβ chain constant region (TRBC) and exhibits excellent internal distribution characteristics when bound to target cells.

[0036] 1.TCR β chain constant region (TRBC) T cell receptors (TCRs) are expressed on the surface of T lymphocytes and are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. When TCRs bind to antigenic 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.

[0037] The TCR is a membrane-bound, disulfide-bonded heterodimer consisting of highly variable alpha (α) and beta (β) chains, typically expressed as part of a complex with an invariant CD3 chain molecule. T cells expressing this receptor are called α:β (or αβ) T cells (about 95% of all T cells). A small number of T cells express an alternative receptor formed by variable gamma (γ) and delta (δ) chains, and are called γδ T cells (about 5% of all T cells).

[0038] Each α-chain and β-chain consists of two extracellular domains: a variable (V) region and a constant (C) region, which are immunoglobulin superfamily (IgSF) domains that form an antiparallel β-sheet. The constant region is close to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. The constant region of the TCR consists of a short connecting sequence in which cysteine ​​residues form disulfide bonds, creating a link between the two chains.

[0039] Both the α and β chains of the TCR have three hypervariable or complementarity-determining regions (CDRs). The β chain's variable region has an additional hypervariable region (HV4), but this does not usually come into contact with the antigen and is therefore not considered a CDR.

[0040] 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 via specific cytoplasmic domains that interact with second messenger and adapter molecules after antigen recognition by αβ or γδ. Cell surface expression of the TCR complex is preceded by the assembly of paired subunits, in which both the transmembrane and extracellular domains of TCRα and β, as well as CD3γ and δ, play a role.

[0041] Therefore, the TCR generally consists of a CD3 complex and TCRα and β chains, with the TCRα and β chains comprising a variable region and a constant region (Figure 1).

[0042] The locus (Chr7:q34) that supplies the TCRβ constant region (TRBC) has duplicated during the course of evolution to produce two nearly identical, functionally equivalent genes: TRBC1 and TRBC2 (Figure 2). Each TCR contains either TRBC1 or TRBC2 in a mutually exclusive manner, and thus each αβ T cell expresses either TRBC1 or TRBC2 in a mutually exclusive manner.

[0043] The inventors previously determined that it is possible to distinguish between TRBC1 and TRBC2 despite the similarity between their sequences. The inventors also previously determined that it is possible to discriminate the amino acid sequences of TRBC1 and TRBC2 while they are present in situ on the surface of cells, such as T cells (International Publication No. 2015 / 132598). In addition, a number of antibodies specific for either TRBC1 or TRBC2 have been produced (International Publication No. 2015 / 132598).

[0044] 2. Antibody conjugate In a first aspect, the invention provides an antibody conjugate that specifically binds to a TCRβ constant region (TRBC), hereinafter "the antibody conjugate of the invention", wherein the antibody has a dissociation rate constant (k -1 ~0.5 seconds -1 ) within the range of d .

[0045] As used herein, the term "antibody conjugate" refers to a compound comprising an antibody conjugated to a therapeutic entity or payload via a chemical linker. When bound to a target antigen on the surface of a cell, the antibody conjugate is internalized and transported to lysosomes, where the payload is released by proteolysis of a cleavable linker (e.g., by cathepsin B found in lysosomes) or, if the payload is bound to the antibody via a non-cleavable linker, by proteolysis of the antibody.

[0046] 2.1. Antibody The antibody conjugate of the present invention comprises an antibody that specifically binds to the TCRβ chain constant region (TRBC).

[0047] As used herein, the term “antibody” refers to a polypeptide having an antigen-binding site comprising at least one complementarity-determining region or CDR. An antibody may contain three CDRs and have an antigen-binding site equivalent to that of a single-domain antibody (dAb), a heavy-chain antibody (VHH), or a nanobody (see Figure 3b). An antibody may contain six CDRs and have an antigen-binding site equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence that provides a suitable scaffold for the antigen-binding site and presents the antigen-binding site in a manner suitable for the antigen to bind to the antigen.

[0048] Full-length antibodies or immunoglobulins typically consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. Each pair of variable regions of light and heavy chains is characterized by the same general structure, which consists of a relatively conserved region called a framework (FR) linked by three hypervariable regions called complementarity-determining regions (CDRs) (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5). thEd., NIH Publication No. 91-3242, Bethesda, MD.; Chothia & Lesk, 1987, J Mol Biol 196:901-17). As used herein, the terms “complementarity-determining region” or “CDR” refer to a region within an antibody that complements the shape of an antigen. Thus, CDRs determine the affinity and specificity of a protein to a particular antigen. The CDRs of the two strands of each pair are aligned by a framework region and acquire the function of binding to a specific epitope. As a result, in the case of VH and VL domains, both the heavy and light chains are characterized by three CDRs, CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3, respectively.

[0049] Several definitions of CDR are commonly used. Kabat's definition, based on sequence variability, is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). Alternatively, the ImMunoGeneTics information system (IMGT) can be used (see http: / / www.imgt.org). According to this system, the complementarity determination region (CDR-IMGT) is a loop region of variability domains delimited according to IMGT-specific numbering for V domains. Within the variability domains, there are three CDR-IMGTs: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C''), and CDR3-IMGT (loop FG). Other definitions of CDR, such as Chothia, AbM, and contact definitions, have also been developed (see http: / / www.imgt.org).

[0050] The antibody conjugate of the present invention may include a full-length antibody or its antigen-binding fragment.

[0051] The antibody conjugate of the present invention may contain a full-length antibody. The full-length antibody may be IgG, IgM, IgA, IgD, or IgE. The full-length antibody may be IgG or IgM.

[0052] The terms “antibody fragment,” “antigen-binding fragment,” “functional fragment of an antibody,” and “antigen-binding moiety” are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Antibody fragments may include, for example, one or more CDRs, variable regions (or parts thereof), constant regions (or parts thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, Fab fragments, F(ab')2 fragments, Fv fragments, single-chain Fv(scFv), domain antibodies (dAb or VH), single-domain antibodies (sdAb), VHH, nanobodies, diabodies, triabodies, trimerbodies, and monobodies.

[0053] The antibody conjugates of the present invention may include antigen-binding domains based on non-immunoglobulin scaffolds. These antibody-binding domains are also called antibody mimes. Non-limiting examples of non-immunoglobulin antigen-binding domains include afibodies, fibronectin artificial antibody scaffolds, anticalin, affilin, DARPin, VNAR, iBody, affimer, finomer, abdulin / nanoantibody, centinlin, alphabodies, nanophytin, and D domains.

[0054] Antibodies can be bifunctional.

[0055] Antibodies may be non-human, e.g., from mice, rats, or camels, chimeric, humanized, or fully human. Antibodies may be synthetic.

[0056] Various antibody formats and modifications currently known in the art or to be developed in the future, used to extend half-life and / or enhance drug delivery, also form part of the present invention.

[0057] The antibody for use in the antibody conjugate of the present invention specifically binds to TRBCs. The ability of the antigen-binding domain to specifically bind to TRBCs can be determined by a number of assays available in the art. Preferably, the binding specificity of the antigen-binding domain is determined by in vitro binding assays such as surface plasmon resonance (SPR), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive ELISA; by immunofluorescence techniques such as immunohistochemistry (IHC), fluorescence microscopy, and flow cytometry; or by immunoprecipitation.

[0058] The antibody for use in the antibody conjugate of the present invention may conjugate either TRBC1 or TRBC2. A method for determining whether an antibody is specific to TRBC1 or TRBC2 is described in International Publication No. 2015 / 132598.

[0059] Antibodies or antibody fragments can deliver various loads to target cells expressing TRBCs. The effectiveness of antibody-drug conjugates (ADCs) depends not only on their binding affinity and specificity to the antigen, but also on their internal distribution. Using TRBC-specific antibodies with different binding affinities, we determined that the kinetic rate constant of the TRBC-specific antibody conjugate is a factor that determines the internal distribution characteristics of the TRBC-specific antibody conjugate upon binding to target cells. Surprisingly, the best internal distribution is obtained using antibodies with rapid dissociation. Furthermore, we were able to determine the binding affinity that exhibits optimal internal distribution characteristics on T cells.

[0060] The terms “internal transfer” or “cellular uptake” are used interchangeably in the context of this invention and, as used herein, refer to the process also known as receptor-mediated endocytosis. Endocytosis is a cellular process in which molecules or substances are transported into a cell via cell membrane encapsulation. After internal transfer, antibody conjugate molecules are transported to lysosomes, where the conjugate is released. Therefore, the rate and extent of antibody conjugate internal transfer are critical to the efficacy of the antibody conjugate.

[0061] Numerous techniques, including microscopy and flow cytometry, can be used to identify antibodies with a desired rate of cell uptake. Co-localization with endosomal proteins based on immunofluorescence microscopy is also used to monitor cell uptake. Unlike immunofluorescence microscopy, flow cytometry using fluorescently labeled antibodies allows for a more rapid and quantitative assessment of antibody internal migration and potentially higher throughput. In flow cytometry-based assays, fluorescently labeled secondary antibodies can be used to measure how much antibody remains surface-bound after the incubation period. Alternatively, cells can be treated with a fluorescently labeled primary antibody before cell surface fluorescence quenching with an anti-fluorophore antibody. Given that ADCs require endocytosis and subsequent acidification to be effective, pH-sensitive labeling is very useful for tracking the internal migration of antibody conjugates. Non-limiting examples of pH-sensitive dyes include fluorescein, which exhibits bright fluorescence that is quenched as pH decreases, and pHrodo dyes, which show very low fluorescence at neutral pH and increase fluorescence as pH becomes more acidic. Methods for labeling antibodies with fluorescent dyes are well known in this field.

[0062] As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and its epitope. Affinity is typically expressed as the dissociation rate constant (k) between the antibody and the antigen. d or k off ) and the association rate constant (k aor k on The ratio of ) that is, k d / k a or k off / k on It is measured as the equilibrium dissociation constant (KD). KD and affinity have an inverse relationship. The terms "association rate constant" or "on velocity" used herein are used in this specification. a " or "k on The term "dissociation rate constant" or "off-rate" or "k" as used herein refers to a constant used to characterize how quickly an antibody binds to its target. d " or "k off The term "KD" refers to a constant used to characterize how quickly an antibody binds to its target. KD is measured in M, and k a is M -1 seconds -1 Measured at k d is seconds -1 It is measured at [location / location].

[0063] The affinity of an antigen-binding domain to any given antigen can be quantified using any conventional method, including, but not limited to, label-dependent methods such as direct and indirect ELISA and radioimmunoassays, as well as label-free methods that enable direct detection and measurement of real-time interactions, such as surface plasmon resonance (SPR) and biolayer interference.

[0064] The KD and kinetic rate constant of the antibody used in the antibody conjugate of the present invention can be measured by SPR. The assay can be performed using different parameters and various commercially available instruments. For example, the KD and kinetic rate constant can be measured using a Biacore T200 instrument at a flow rate of 30 ml / min at 25°C, with HBSP1 as the running and dilution buffer (GE Healthcare BioSciences). The kinetic rate constant can be obtained by curve fitting following a 1:1 Langmuir binding model.

[0065] The inventors have found that the antibody takes 0.001 seconds-1 ~0.3 seconds -1 Dissociation rate constant k within the range d It was determined that the optimal internal migration characteristics are exhibited when the following is present. The antibody for use in the antibody conjugate of the present invention is 0.001 seconds -1 ~0.25 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.001 seconds -1 ~0.2 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.001 seconds -1 ~0.15 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.001 seconds -1 ~0.1 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.002 seconds -1 ~0.3 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.003 seconds -1 ~0.3 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.004 seconds -1 ~0.3 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.005 seconds -1 ~0.3 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.006 seconds -1 ~0.3 seconds -1 k within the range d It may have. The antibody for use in the antibody conjugate of the present invention is 0.007 seconds -1 ~0.3 seconds -1 k within the range dmay have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.008 seconds -1 to 0.3 seconds -1 may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.009 seconds d to 0.3 seconds -1 may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.01 seconds -1 to 0.3 seconds d may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.01 seconds -1 to 0.2 seconds -1 may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.01 seconds d to 0.15 seconds -1 may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.01 seconds -1 to 0.1 seconds d may have. The antibody for use in the antibody conjugate of the present invention has a k of from 0.002 seconds -1 to 0.1 seconds -1 may have. The antibody conjugate according to claim 1, wherein the antibody has a k in the range of 0.002 seconds d to 0.1 seconds -1 may have. The antibody conjugate according to claim 1, wherein the antibody has a k in the range of 0.017 seconds -1 to 0.083 seconds d may have. The antibody conjugate according to claim 1, wherein the antibody has a k in the range of 0.017 seconds -1 to 0.083 seconds -1 may have. The antibody conjugate according to claim 1, wherein the antibody has a k in the range of 0.017 seconds d to 0.083 seconds -1 to 0.083 seconds -1 may have. The antibody conjugate according to claim 1, wherein the antibody has a k in the range of 0.017 seconds d to 0.083 seconds

[0066] The antibody for use in the antibody conjugate of the present invention may further have an association rate constant (k 2 M -1 ) in the range of 1×10 6 M -1 to 1×10 a M 2 M -1 The antibody for use in the antibody conjugate of the present invention may further have a k in the range of 5×10<于 5 M<于 -1 to 5×10<于 amay further have. The antibody for use in the antibody conjugate of the present invention has a k within the range of 1×10 3 M -1 ~5×10 5 M -1 and may further have. The antibody for use in the antibody conjugate of the present invention has a k within the range of 5×10 3 M -1 ~5×10 5 M -1 and may further have. The antibody for use in the antibody conjugate of the present invention has a k within the range of 1×10 4 M -1 ~1×10 5 M -1 and may further have. The antibody for use in the antibody conjugate of the present invention has a k within the range of 5×10 4 M -1 ~1×10 5 M -1 and may further have. The antibody for use in the antibody conjugate of the present invention has a k within the range of 5×10 a and may further have.

[0067] The antibody for use in the antibody conjugate of the present invention may further have an affinity constant (KD) within the range of 1×10 ー10 M to 1×10 ー5 M. The antibody for use in the antibody conjugate of the present invention may further have a KD within the range of 1×10 -10 M to 5×10 -6 M. The antibody for use in the antibody conjugate of the present invention may further have a KD within the range of 5×10 -10 M to 1×10 -6 M. The antibody for use in the antibody conjugate of the present invention may further have a KD within the range of 5×10 -10 M to 5×10 -7 M. The antibody for use in the antibody conjugate of the present invention may further have a KD within the range of 1×10 -9 M to 5×10 -7 M. The antibody for use in the antibody conjugate of the present invention may further have a KD within the range of 5×10 -9 M to 1×10 -7 ​​​​​​It may have further KDs within the range of M.

[0068] The antibody for use in the antibody conjugate of the present invention is 0.001 seconds -1 ~0.35 seconds -1 k within the range d , 1 x 10 2 M -1 ~1 × 10 6 M -1 k within the range a and 1 × 10 -10 M~1×10 -5 It may have KD in the range of M.

[0069] The antibody used in the antibody conjugate of the present invention may exhibit increased internal migration upon binding to target cells compared to the internal migration of 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.

[0070] The internal migration of the antibody conjugate of the present invention upon binding to target cells may be increased compared to the internal migration of the reference antibody. The internal migration of the antibody conjugate upon binding to target cells may be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, or at least 10,000% compared to the internal migration of the reference antibody. Methods for determining and quantifying the internal migration of antibody conjugates have been previously described in detail.

[0071] The antibody for use in the antibody conjugate of the present invention can specifically bind to TRBC1.

[0072] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC1, has the following mutations or combinations of mutations 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 -G106A in the VH domain; -Y32F in the VH domain; - G31S in the VH domain; - G26P and T28K in the VH domain; -Y102M in VH domain It may include one of the following.

[0073] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC1, comprises a VH domain having the sequence shown in SEQ ID NO: 1, a VL domain having the sequence shown in SEQ ID NO: 2, and the following mutations or combinations of mutations: -G106A in the VH domain; -Y32F in the VH domain; - G31S in the VH domain; - G26P and T28K in the VH domain; -Y102M in the VH domain; -T28K, Y32F, A100N and N103L in the VH domain, and N35K in the VL domain; -T28K, Y32F and A100N in the VH domain, and N35K in the VL domain; or -T28K, Y32F, A100N and A107S in the VH domain It can consist of one of the following.

[0074] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC1, has the following mutations 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: - G106A in the VH domain It may include.

[0075] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC1, comprises a VH domain having the sequence shown in SEQ ID NO: 1, a VL domain having the sequence shown in SEQ ID NO: 2, and the following mutations: - G106A in the VH domain It can consist of.

[0076] These specific combined mutations were shown to alter TRBC1 binding in a manner useful for ADC therapeutic strategies (see Table 1). [Table 1-1] [Table 1-2]

[0077] The antibody for use in the antibody conjugate of the present invention is capable of specifically binding to TRBC2.

[0078] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC2, has the following combination of mutations 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: -T28K, Y32F, A100N, Y102L and N103M in the VH domain, and N35R in the VL domain; -T28K, Y32F, and A100N in the VH domain; or -T28R, Y32F and A100N in the VH domain It may include one of the following.

[0079] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC2, comprises a VH domain having the sequence shown in SEQ ID NO: 1, a VL domain having the sequence shown in SEQ ID NO: 2, and the following mutations: -T28K, Y32F, A100N, Y102L and N103M in the VH domain, and N35R in the VL domain; -T28K, Y32F, and A100N in the VH domain; or -T28R, Y32F and A100N in the VH domain It can consist of one of the following.

[0080] These specific combined mutations were shown to alter TRBC2 binding in a manner useful for ADC therapeutic strategies (see Table 2). [Table 2]

[0081] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC2, has the following mutations 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: -T28K, Y32F, A100N and N103L in the VH domain, and N35K in the VL domain It may include.

[0082] The antibody for use in the antibody conjugate of the present invention, which is specific to TRBC2, comprises a VH domain having the sequence shown in SEQ ID NO: 1, a VL domain having the sequence shown in SEQ ID NO: 2, and the following mutations: -T28K, Y32F, A100N and N103L in the VH domain, and N35K in the VL domain It can consist of.

[0083] As used herein, the term “reference antibody” refers to a humanized JOVI-1 antibody, i.e., hJOVI-1, comprising 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 has been previously disclosed by Viney et al. (Viney et al., 1992, Hybridoma, 11:701-13). [ka] [ka]

[0084] The antibody for use in the antibody conjugate of the present invention may include a VH domain having the sequence shown in Sequence ID No. 1 and a VL domain having the sequence shown in Sequence ID No. 2.

[0085] Mutants or variants of antibodies for use in antibody conjugates of the present invention, which maintain their specificity and internal migration characteristics, also form part of the present invention. As used herein, the terms “mutant” or “variant” refer to polypeptides that differ from the specifically listed polypeptides, i.e., reference or parent polypeptides, by amino acid insertions, deletions, and / or substitutions, for example, using recombinant DNA technology or by de novo synthesis. Mutants and variants are used without distinction in the context of the present invention.

[0086] 2.2. Therapeutic entity or mounted device The antibody conjugate of the present invention comprises an antibody bound to a therapeutic entity or payload. For the purpose of investigating the internal migration characteristics of the antibody conjugate, the payload may be replaced with a detection entity.

[0087] As used herein, the terms “therapeutic entity” or “carryover” refer to any molecule that inhibits or suppresses cellular function and / or causes cellular destruction (cell death) and / or exerts an antiproliferative effect. Carryovers may be drugs or radionuclides.

[0088] The antibody in the antibody conjugate of the present invention may be conjugated with a chemotherapeutic entity, a radionuclide, or a detection entity.

[0089] As used herein, the term “chemotherapeutic entity” refers to any molecule that inhibits or suppresses cellular function and / or induces cell death and / or exerts an antiproliferative effect. Hereinafter, the resulting conjugate will be referred to as the “antibody-drug conjugate (ADC) of the present invention.” The chemotherapeutic entity may be a cytotoxic drug or a cytotoxin. Numerous classes of cytotoxic agents, including but not limited to tubulin inhibitors, DNA damage agents, topoisomerase I inhibitors and RNA polymerase II inhibitors, are known in the art to have potential utility in antibody molecules and can be used in the ADCs described herein.

[0090] To date, microtubules have five known binding sites: vinca alkaloid binding sites, taxane binding sites, colchicine binding sites, mytansine binding sites, and laurymaride binding sites. Microtubule / tubulin inhibitors can be classified into two main categories according to their mechanism of action: those that promote tubulin polymerization and stabilize microtubule structure (substances that bind to taxane and laurymaride binding sites) and those that inhibit tubulin polymerization and destabilize microtubule structure (substances that bind to vinca alkaloid binding sites, mytansine binding sites, and colchicine binding sites). Non-exclusive examples of tubulin inhibitors that bind to the vinca alkaloid binding site include vincristine, vinblastine, vinflunin, halichondrin B, eribulin mesylate, cryptophycin, and drastatin, e.g., auristatin MMAF, MMAE, PF-06390101, MMAD, auristatin E, auristatin W analogues, auristatin f-HPA, amberstatin 269, and AGD-0182. Non-exclusive examples of tubulin inhibitors that bind to the mytansine binding site include mytansine and mytansinoids, e.g., DM1 and DM4. Non-exclusive examples of tubulin inhibitors that bind to the colchicine binding site include colchicine, 2-methoxyestradiol, sulfonamides, and aspergillus derivatives. Non-exclusive examples of tubulin inhibitors that bind to the taxane binding site include paclitaxel, docetaxel, cyclostreptin, eryuterobin, ABI-007, ixabepyrone, patupyrone, and BMS-310705. Non-exclusive examples of tubulin inhibitors that bind to the laurimalide binding site include laurimalide and perolside A.

[0091] DNA damaging agents include, but are not limited to, calicheamicin (ozogamicin), e.g., calicheamicin γ1; pyrrolobenzodiazepines, e.g., PDB-Talilin and SG3249; duocalmycin, e.g., DUBA; camptothecin analogs, e.g., SN38 and DX-8951; anthracyclines; and doxorubicin (adriamycin). Alkylating agents also include nitrosourea, ethyleneimine / methylmelamine, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophyllotoxin, platinum-coordinated complexes such as cisplatin, and carboplatin enzymes such as L-asparaginase.

[0092] Topoisomerase I inhibitors include, but are not limited to, irinotecan, topotecan, and camptothecin.

[0093] RNA polymerase II inhibitors include, but are not limited to, amatoxins such as α-amanitin.

[0094] Cytotoxins suitable for use in ADCs are also described, for example, in International Publication No. 2015 / 155345 and International Publication No. 2015 / 157592.

[0095] Chemotherapy agents include biological response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; adrenal cortical inhibitors such as anthracendione, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, mitotane (o,p'-DDD), and aminoglutethimide; hormones and antagonists including adrenal cortical steroid antagonists such as prednisone and its equivalents, dexamethasone, and aminoglutethimide; and capron These may include progestins such as hydroxyprogesterone acid, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens such as flutamide.

[0096] The antibody in the antibody conjugate of the present invention can be conjugated with a radionuclide. Hereinafter, the resulting conjugate will be referred to as the “antibody-radionuclide conjugate of the present invention (ARC)”. Radioimmune complexes have unique theranotic (i.e., therapeutic and diagnostic) potential. For diagnostic purposes, the antibody can be labeled with a radionuclide that is compatible with imaging procedures such as single-photon emission computed tomography or positron emission tomography (PET). For therapeutic purposes, the selection of the radionuclide depends largely on the size of the tumor to be treated. 90 High-energy beta emitters such as Y are suitable for treating larger tumors. 131 I and 177Medium-energy beta emitters such as Lu are more effective in treating smaller tumors. Radionuclides suitable for use in ARC are well known in the art, and other radionuclides are also intended in this invention. One of the main attractive features of radioimmunotherapy is the crossfire or bystander effect, i.e., the ability to damage cells adjacent to the antibody localization site. In most cases, radiolabeling of antibodies is achieved by iodation of tyrosine or by conjugation of the antibody molecule with a metal chelating agent such as diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0097] The detectable entities may be fluorescent entities, such as fluorescent peptides or dyes or labels. As used herein, the term “fluorescent entity” refers to a part that emits light at a detectable wavelength after excitation. Examples of fluorescent entities 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. pH-sensitive dyes or labels are particularly advantageous for tracking the internal migration of antibody conjugates. Non-limiting examples of pH-sensitive dyes include fluorescein, which exhibits bright fluorescence that is quenched as the pH decreases, and pHrodo dyes, which exhibit very low fluorescence at neutral pH and increase fluorescence as the pH becomes more acidic.

[0098] The antibody conjugate of the present invention, conjugated to a detectable entity, can be used to determine the TRBCs of malignant T cells.

[0099] The antibody conjugate of the present invention may contain at least one therapeutic molecule or loading molecule (including a detection entity) conjugated thereto. The antibody conjugate of the present invention may contain any appropriate number. To achieve a desired therapeutic effect, the antibody conjugate of the present invention may contain, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more loading molecules conjugated thereto.

[0100] 2.3. Conjugation Chemistry The antibody conjugate of the present invention comprises an antibody that is conjugated to a chemotherapeutic entity, radionuclide, or detection entity via conjugation or a chemical linker.

[0101] Traditional chemical conjugation of loaders onto antibodies occurs via solvent-exposed lysine residues (via succinimide esterification) or via interchain cysteine ​​residues (maleimide chemistry). Alternatively, various approaches have been developed to obtain site-specific conjugations in which the attachment site of the cytotoxic agent to the antibody is precisely defined. These techniques include: (a) applying engineered cysteines such as THIOMAB to enable site-specific conjugation only on the heavy chain of the antibody; (b) introducing non-natural amino acids into proteins and antibodies through mutagenesis, such as incorporating p-acetylphenylalanine or selenocysteine ​​into IgG1; (c) using enzymatic and chemioenzymatic methods to generate site-specific conjugations, such as using engineered glycotransferases, transglutaminases, transpeptidase-soltases or formylglycine-producing enzymes to form site-specific functionalization of the antibody; and (d) using tubulin tagging strategies.

[0102] If the antibody is a complete antibody or a monoclonal antibody, the payload can be chemically conjugated to the side chain of an amino acid such as cysteine ​​at a specific Kabat position in the Fc region of the antibody. Positions on the Fc region suitable for site-directed conjugation are well known in the art. Alternatively, the payload can be conjugated to the antibody via thiol-maleimide bonds in the hinge and heavy / light chains.

[0103] Chemical linkers must be sufficiently stable in systemic circulation and capable of rapidly and efficiently releasing cytotoxic agents during internal translocation of antibody conjugates within cancer cells. According to drug release mechanisms, linkers for antibody conjugates are generally classified into cleavable linkers (acid-unstable linkers, protease-cleavable linkers, and disulfide linkers) and non-cleavable linkers (Table 3). Antibody conjugates with non-cleavable linkers require lysosomal degradation of the antibody to release cytotoxic agents, while antibody conjugates with cleavable linkers involve hydrolysis, enzymatic reactions, or reduction to selectively release cytotoxic drugs based on the physiological environment to which the antibody conjugate is exposed. [Table 3]

[0104] Three main types of cleavable linkers—acid-cleavable linkers, protease-cleavable linkers, and disulfide linkers—are frequently used in ADCs. Acid-cleavable linkers, such as hydrazine linkers (AcBut), are designed to be stable at the neutral pH of circulation but can be hydrolyzed within lysosomes, which have a low pH environment. Protease-cleavable linkers are also used to keep antibody conjugates intact in systemic circulation and to allow for the easy release of cytotoxic drugs from the antibody conjugate by lysosomal enzymes within cancer cells. For example, valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-Lys) provide excellent stability and PK / PD profiles to antibody conjugates. Furthermore, antibody conjugates with disulfide linkers such as N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) and N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) utilize reduced glutathione, which has high intracellular concentrations, to release free drugs into cells. Antibody conjugates with reducible disulfide linkers generate uncharged metabolites that can diffuse into adjacent cells and induce bystander death, which is advantageous for killing heterogeneous tumors.

[0105] A non-limiting example of a non-cleavage linker is the thioether linker (N-succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate [MCC]).

[0106] Other linkers include hydrophilic linkers such as β-glucuronide linkers, Sulfo-SPDB, and Mal-PEG4-NHS.

[0107] 3. Pharmaceutical composition In another aspect, the present invention also relates to a pharmaceutical composition containing the antibody conjugate of the present invention, hereinafter referred to as "the pharmaceutical composition of the present invention."

[0108] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

[0109] The term "antibody conjugate of the present invention" is described in detail in relation to the aforementioned aspects of the present invention, and its features and embodiments are equally applicable to these aspects of the present invention.

[0110] Administration The administration of the antibody conjugate of the present invention can be achieved using any of the various routes that make the active ingredient bioavailable. For example, the active ingredient can be administered via oral and parenteral routes, intraperitoneal, intravenous, subcutaneous, percutaneous, intramuscular, or local delivery, such as by catheter or stent.

[0111] Typically, a physician determines the most appropriate actual dosage for each individual patient, and this dosage varies depending on the patient's age, weight, and response. The dosage is sufficient to reduce or deplete the number of malignant clonal T cells.

[0112] 4. Treatment Method In another aspect, the present invention provides an antibody conjugate for use in pharmaceuticals.

[0113] In another aspect, the present invention provides a method for treating T-cell lymphoma or leukemia in a subject, comprising the step of administering the antibody conjugate of the present invention to a subject that requires administration of the antibody conjugate of the present invention, hereinafter referred to as the "method of treatment of the present invention." The administration step may be in the form of the above-described pharmaceutical composition.

[0114] This aspect of the present invention can, alternatively, be formulated as an antibody conjugate of the present invention for use in the treatment of T-cell lymphoma or leukemia, hereinafter referred to as "Antibody Conjugate for Use of the Present Invention."

[0115] This aspect of the present invention can, alternatively, be formulated as the use of the antibody conjugate of the present invention in the manufacture of a pharmaceutical product for treating T-cell lymphoma or leukemia.

[0116] The term "antibody conjugate of the present invention" is described in detail in relation to the aforementioned aspects of the present invention, and its features and embodiments are equally applicable to these aspects of the present invention.

[0117] The method for treating T-cell lymphoma and / or leukemia relates to the therapeutic use of the antibody conjugate of the present invention, which can be administered to subjects having a pre-existing T-cell lymphoma and / or leukemia, in order 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.

[0118] Methods for preventing T-cell lymphoma and / or leukemia relate to the prophylactic use of antibody conjugates of the present invention. Such antibody conjugates may be administered to subjects who are not yet diagnosed with T-cell lymphoma and / or leukemia, and / or who do not exhibit any symptoms of T-cell lymphoma and / or leukemia, in order to suppress or reduce the cause of the disease, or to mitigate or prevent the development of at least one symptom associated with the disease. Subjects may be predisposed to T-cell lymphoma and / or leukemia, or may be considered to be at risk of developing T-cell lymphoma and / or leukemia.

[0119] These therapeutic applications involve administering a therapeutically effective amount of the antibody conjugate of the present invention.

[0120] Treatment of T-cell lymphoma or leukemia in a subject may include administering an antibody conjugate to the subject to cause selective depletion of malignant T cells, along with normal T cells that express the same TRBCs as malignant T cells, but not to cause depletion of normal T cells that express TRBCs not expressed by malignant T cells.

[0121] As used in the context of this invention, the terms "subject" or "individual" refer to a member of a mammalian species, preferably a human being of any age or race, male or female.

[0122] The method may also include a step of examining the TCRβ chain constant region (TCRB) of malignant T cells derived from the target to determine whether the malignant T cells express TRBC1 or TRBC2. This information assists healthcare professionals in determining the appropriate TRBC selectivity of the antibody conjugate to be administered.

[0123] As used herein, the term “therapeutic dose” refers to the amount of the antibody conjugate of the present invention required to achieve recognizable prevention, cure, delay, reduction or improvement of the severity of one or more symptoms of T-cell lymphoma and / or leukemia.

[0124] The method of the present invention may be used to treat T cells. The term "lymphoma" is used herein in accordance with its standard meaning to refer to cancer that typically occurs in the lymph nodes but may also affect the spleen, bone marrow, blood, and other organs. Lymphoma typically presents as a solid tumor of lymphoid cells. The main symptom associated with lymphoma is lymphadenopathy, but secondary (B) symptoms may include fever, night sweats, weight loss, loss of appetite, fatigue, respiratory distress, and itching.

[0125] The method of the present invention may be used to treat T-cell leukemia. “Leukemia” is used herein in accordance with its standard meaning to refer to cancer of the blood or bone marrow.

[0126] The following is an illustrative and non-exclusive list of diseases that can be treated by the methods of the present invention.

[0127] Peripheral T-cell lymphoma Peripheral T-cell lymphomas are relatively rare lymphomas, accounting for less than 10% of all non-Hodgkin lymphomas (NHLs). However, peripheral T-cell lymphomas have an invasive clinical course, and the causes and precise cellular origins of most T-cell lymphomas are still not clearly defined.

[0128] Lymphoma usually first presents as swelling in the neck, armpit, or groin. Further swelling may occur if other lymph nodes, such as those in the spleen, are located nearby. Generally, enlarged lymph nodes can invade blood vessels, nerves, or gastric spaces, 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.

[0129] To describe a prognostic and therapeutically meaningful classification of peripheral T-cell lymphomas, the WHO classification utilizes morphological and immunophenotypic features in conjunction with clinical aspects and, in some cases, genetics (Swerdlow et al.; WHO classification of tumors of haematopoietic and lymphoid tissues. 4th ed.; Lyon: IARC Press; 2008). The anatomical localization of neoplastic T cells is partially similar 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 of the findings of T-cell lymphomas, including the cellular distribution of T-cell lymphomas, several aspects of morphology, and even related clinical findings.

[0130] The most common type of T-cell lymphoma is peripheral T-cell lymphoma, unspecified type (PTCL-NOS), which accounts for 25% of all cases, followed by angioimmunoblastic T-cell lymphoma (AITL) (18.5%).

[0131] Peripheral T-cell lymphoma, nonspecific (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. PTCL-NOS is diagnosed by exclusion and does not correspond to any of the specific mature T-cell lymphoma entities listed in the current WHO 2008. Therefore, PTCL-NOS is similar to diffuse large B-cell lymphoma, unspecified type (DLBCL-NOS).

[0132] Most patients are adults, with a median age of 60 years and a male-to-female ratio of 2:1. The majority of cases originate from the lymph nodes, but extra-lymphatic symptoms occur in about 13% of patients, most commonly metastasizing to the skin and gastrointestinal tract.

[0133] The cytological spectrum is very broad, ranging from pleomorphic to monomorphic. Three morphologically defined variants have been described, including the lymphoepithelioid (Rennert) variant, the T-zone variant, and the follicular variant. The lymphoepithelioid variant of PTCL contains abundant background epithelial-like histiocytes and is generally CD8-positive. The lymphoepithelioid variant of PTCL is associated with a better prognosis. The follicular variant of PTCL-NOS is emerging as a potentially distinct clinicopathological entity.

[0134] The majority of PTCL-NOS cases exhibit a mature T-cell phenotype and are almost always CD4-positive. 75% of cases show variable loss of at least one pan-T-cell marker (CD3, CD2, CD5, or CD7), with CD7 and CD5 being downregulated in most cases. CD30 and rarely CD15 may be expressed, with CD15 being an adverse prognostic feature. CD56 expression also has a rare but negative prognostic impact. Further adverse pathological prognostic factors include proliferation rates greater than 25% based on KI-67 expression and the presence of more than 70% transformed cells. Immunophenotypic analysis of these lymphomas provides little insight into their biology.

[0135] Angioimmunoblastic T-cell lymphoma (AITL) AITL is a systemic disease characterized by pleomorphic infiltrates with lymph nodes, prominent high endothelial venules (HEVs), and perivascular dilation of a follicular dendritic cell (FDC) reticular structure. AITL is considered a de novo T-cell lymphoma that usually originates from follicular helper (TFH) αβ T cells found in germinal centers.

[0136] AITL is the second most common entity among peripheral T-cell lymphomas and NK / T-cell lymphomas, accounting for approximately 18.5% of cases. AITL occurs in middle-aged to elderly adults, with a median age of 65 years, and has nearly equal incidence in men and women. Clinically, patients usually have advanced disease with generalized lymphadenopathy, hepatosplenomegaly, and marked systemic symptoms. Itchy skin rashes are commonly present. Polyclonal hypergammaglobulinemia associated with autoimmune phenomena is often present.

[0137] AITL exhibits three distinct morphological patterns. Early lesions of AITL (Pattern I) typically present with conserved structures featuring characteristic hyperplastic follicles. Neoplastic proliferation is localized around the follicles. In Pattern II, nodular structures are partially absent, with little retention of regressed follicles. The marginal sinuses are preserved and may even be dilated. The paracortex contains branching HEVs, and there is proliferation of FDCs beyond the B-cell follicles. Neoplastic cells are small to medium in size, with minimal cytological atypicality. Neoplastic cells often have clear to pale cytoplasm and may exhibit a distinct T-cell membrane. A pleomorphic inflammatory background is usually evident.

[0138] AITL is a T-cell malignancy, but it is characterized by the expansion and proliferation of B cells and plasma cells, which may reflect the function of neoplastic cells as TFH cells. Both EBV-positive and EBV-negative B cells are present. Occasionally, atypical B cells may resemble Hodgkin / Reed-Sternberg-like cells morphologically and immunophenotypicly, sometimes leading to diagnostic confusion with their actual identity. B cell proliferation in AITL can be widespread, and some patients develop secondary EBV-positive diffuse large B-cell lymphoma (DLBCL) or—more rarely—EBV-negative B-cell tumors, often with plasmacytic differentiation.

[0139] Neoplastic CD4-positive T cells in AITL exhibit strong expression of CD10 and CD279 (PD-1) and are positive for CXCL13. CXCL13 leads to increased B cell recruitment to lymph nodes via attachment to HEVs, B cell activation, plasma cell differentiation, and expansion of the FDC reticular structure, all contributing to the morphological and clinical features of AITL. Marked PD-1 expression in perifollicular tumor cells is particularly useful in distinguishing AITL pattern I from reactive follicular and paracortical hyperplasia.

[0140] The follicular variant of PTCL-NOS is a distinct entity possessing the TFH phenotype. In contrast to AITL, the follicular variant of PTCL-NOS does not exhibit prominent extrafollicular expansion of HEV or FDC reticular structures. Neoplastic cells form intrafollicular aggregates and may present with symptoms similar to B-cell follicular lymphoma, but may also have an interfollicular proliferation pattern or be accompanied by an enlarged mantle. Patients more often present with early disease with partial lymph node metastasis and may lack the systemic symptoms associated with AITL; therefore, clinically, the follicular variant of PTCL-NOS is distinct from AITL.

[0141] Anaplastic large cell lymphoma (ALCL) ALCL can be subdivided into ALCL-anaplastic lymphoma kinase (ALK)+ or ALCL-ALK-.

[0142] ALCL-ALK+ is one of the most distinct entities among peripheral T-cell lymphomas, possessing characteristic "Hallmark cells" with horseshoe-shaped nuclei and expressing ALK and CD30. ALCL-ALK+ accounts for approximately 7% of all peripheral T-cell and NK-cell lymphomas and is most common in the first 30 years of life. Patients often present with lymphadenopathy, but metastasis to extra-nodal sites (skin, bone, soft tissue, lung, liver) and B syndrome are common.

[0143] ALCL and ALK+ exhibit a broad morphological spectrum with five distinct patterns described, although all variants contain some Hallmark cells. Hallmark cells have eccentric horseshoe-shaped or kidney-shaped nuclei and prominent perinuclear eosinophilic Golgi regions. Tumor cells proliferate in an adhesive pattern and are prone to sinus metastasis. In the small cell variant, smaller tumor cells are dominant, while in the lymphohistiocytic variant, abundant histiocytes mask the presence of many small tumor cells.

[0144] By definition, all cases are positive for ALK and CD30, with expression typically weaker in smaller tumor cells. Loss of pan-T cell markers is often present, with 75% of cases lacking CD3 surface expression.

[0145] ALK expression is a result of a characteristic recurrent genetic alteration consisting of rearrangement of the ALK gene on chromosome 2p23 to one of many partner genes, leading to the expression of a chimeric protein. The most common partner gene is nucleophosmin (NPM1) on chromosome 5q35, occurring in 75% of cases, resulting in t(2;5)(p23;q35). The cellular distribution of ALK in different translocation mutants can vary depending on the partner gene.

[0146] ALCL-ALK- is included as a provisional category in the 2008 WHO classification. ALCL-ALK- is defined as a CD30-positive T-cell lymphoma that exhibits an adherent growth pattern and the presence of Hallmark cells, but lacks ALK protein expression and is morphologically indistinguishable from ALCL-ALK+.

[0147] In contrast to ALCL-ALK+, which is more common in children and young adults, patients with ALCL-ALK- are typically adults aged 40–65 years. ALCL-ALK- can be present in both lymph nodes and extranodal tissue, although the latter is less common in ALCL-ALK+. Most cases of ALCL-ALK- show loss of lymph node structure due to sheets of adherent neoplasms with typical "holemark" features. In contrast to ALCL-ALK+, small cell morphology variants are not observed.

[0148] Unlike its ALK+ counterpart, ALCL-ALK- exhibits greater conservation of surface T cell marker expression, but less expression of cytotoxic markers and epithelial membrane antigens (EMAs). Gene expression signatures and repetitive chromosomal imbalances differ between ALCL-ALK- and ALCL-ALK+, confirming that they are distinct entities at the molecular and genetic levels.

[0149] 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 significantly better than the 5-year overall survival rate for PTCL-NOS (32%).

[0150] Enteropathy-associated T-cell lymphoma (EATL) EATL is an invasive neoplasm thought to originate from intraepithelial T cells in the intestine. Two types of EATL, type I (which accounts for the majority of EATL cases) and type II (which accounts for 10-20% of cases), are recognized in the 2008 WHO classification as being morphologically, immunohistochemically, and genetically distinct.

[0151] Type I EATL is usually associated with overt or clinically asymptomatic gluten-irritable bowel syndrome and is more common in patients of Northern European descent due to the high prevalence of celiac disease in the Northern European population.

[0152] Most commonly, EATL lesions are found in the jejunum or ileum (90% of cases), and rarely in the duodenum, colon, stomach, or the outer regions of the gastrointestinal tract. Intestinal lesions are usually multifocal with mucosal ulcers. The clinical course of EATL is invasive, and most patients die from the disease or its complications within one year.

[0153] The cytological spectrum of EATL type I is broad, and some cases may contain undifferentiated cells. In some cases, there is a pleomorphic inflammatory background that can mask the neoplastic component. The intestinal mucosa in the area adjacent to the tumor often exhibits features of celiac disease, with blunted villi and an increased number of intraepithelial lymphocytes (IELs) that may correspond to lesion progenitor cells.

[0154] Immunohistochemistry reveals that neoplastic cells are often CD3+CD4-CD8-CD7+CD5-CD56-βF1+ and contain cytotoxic granule-related proteins (TIA-1, granzyme B, perforin). CD30 is partially expressed in almost all cases. In EATL, CD103, a mucosal homing receptor, may be expressed.

[0155] Type II EATL, also known as monomorphic CD56+ intestinal T-cell lymphoma, is defined as an intestinal tumor composed of small to medium-sized monomorphic T cells expressing both CD8 and CD56. Lateral spread of the tumor is present within the mucosa, and there is often no inflammatory background. Most cases express γδTCRs, but some cases are associated with αβTCRs.

[0156] Type II EATL is more widely distributed globally than Type I EATL and is often found in Asian or Latin American populations where celiac disease is rare. In individuals of European descent with EATL, Type II accounts for approximately 20% of intestinal T-cell lymphomas, and at least a portion of these cases have a history of celiac disease. The clinical course is invasive.

[0157] Hepatosplenic T-cell lymphoma (HSTL) HSTL is generally an invasive systemic neoplasm originating from γδ cytotoxic T cells of the innate immune system, but in rare cases it can also originate from αβ T cells. HSTL is one of the rarest T-cell lymphomas, typically affecting adolescents and young adults (median age 35 years), and is strongly male-dominant.

[0158] 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. Most cases originate from NK cells, but some cases originate from cytotoxic T cells. Extranodal NK / T-cell lymphoma, nasal type, is universally associated with Epstein-Barr virus (EBV).

[0159] Cutaneous T-cell lymphoma The method of the present invention may also be used to treat cutaneous T-cell lymphoma.

[0160] Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells into the skin, which manifests as a variety of lesions. These lesions change shape as the disease progresses, typically beginning as a rash-like appearance and eventually forming plaques and tumors before metastasizing to other parts of the body.

[0161] Cutaneous T-cell lymphomas include those mentioned in the following exemplary, non-exclusive list: mycosis fungoides, Paget's reticular dread, 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 cell T-cell lymphoma, pleomorphic T-cell lymphoma, Rennard lymphoma, subcutaneous T-cell lymphoma, and vascular central lymphoma.

[0162] The signs and symptoms of CTCL vary depending on the specific disease, the two most common types being mycosis fungoides and Sézary syndrome. Classical mycosis fungoides is divided into three stages. - Erythema (atrophic or non-atrophic): Nonspecific dermatitis, erythema of the lower trunk and buttocks; minimal / absent pruritus; -Plagues: Severely itchy plaques, lymphadenopathy; and Tumor: Tendency to form ulcers

[0163] 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.

[0164] Of all primary cutaneous lymphomas, 65% are T-cell lymphomas. The most common immunophenotype is CD4-positive. Because the term cutaneous T-cell lymphoma encompasses a wide variety of disorders, there is no common pathophysiology for these diseases.

[0165] The main pathogenesis of cutaneous T-cell lymphoma (i.e., mycosis fungoides) is not yet understood. Mycosis fungoides may precede a T-cell mediated chronic inflammatory skin disease, which can occasionally progress to a fatal lymphoma.

[0166] Primary cutaneous ALCL (C-ALCL) C-ALCL is often indistinguishable from ALC-ALK- depending on its morphology. C-ALCL is defined as a large cell cutaneous tumor with an undifferentiated, pleomorphic, or immunoblastic morphology in which more than 75% of cells express CD30. Along with lymphomatoid papulosis (LyP), C-ALCL belongs to the range of primary cutaneous CD30-positive T-cell lymphoproliferative disorders, forming the second most common group of post-mycosis cutaneous T-cell lymphoproliferations.

[0167] The immunohistochemical staining profile is very similar to that of ALCL-ALK-, with a higher proportion of cases staining positively for cytotoxic markers. At least 75% of tumor cells should be positive for CD30. CD15 may also be expressed, and if lymph node metastasis occurs, differentiation from classical Hodgkin lymphoma can be difficult. Rare cases of ALCL-ALK+ may present with focal skin lesions that resemble C-ALCL.

[0168] T-cell acute lymphoblastic leukemia T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% and 25% of ALL cases in the pediatric and adult cohorts, respectively. Patients typically have high white blood cell counts and may present with organ enlargement, particularly mediastinal enlargement and central nervous system (CNS) infiltration.

[0169] The method of the present invention may be used to treat T-ALL associated with malignant T cells expressing TCRs containing TRBCs.

[0170] T-cell prolymphocytic leukemia T-cell prelymphocytic leukemia (T-PLL) is a mature T-cell leukemia that exhibits invasive behavior and a tendency to metastasize to the blood, bone marrow, lymph nodes, liver, spleen, and skin. T-PLL primarily develops in 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.

[0171] In peripheral blood, T-PLL consists of medium-sized lymphocytes with a single nucleus and basophilic cytoplasm that occasionally has vesicles or projections. The nuclei are usually round to oval in shape, and occasionally patients have cells with more irregular nuclear contours that resemble the gyriform nuclear shape seen in Sézary syndrome. The small cell variant accounts for 20% of all T-PLL cases, while the Sézary cell-like (gyriform) variant is seen in 5% of cases.

[0172] T-PLL is characterized by an immunophenotype of mature (postthymic) T lymphocytes, with neoplasms typically 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%.

[0173] T-cell lymphomas or leukemias that should be treated or prevented may be selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); anaplastic large cell lymphoma (ALCL); enteropathy-associated T-cell lymphoma (EATL); hepatosplenic T-cell lymphoma (HSTL); extranodal NK / T-cell lymphoma nasal type; cutaneous T-cell lymphoma; primary cutaneous ALCL; T-cell prolymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0174] The treatment method may include the step of administering the antibody conjugate of the present invention. Those skilled in the art will be able to determine by conventional methods the amount of the antibody conjugate of the present invention that can exert a therapeutic effect on a patient.

[0175] In another aspect, the present invention relates to an antibody conjugate for use in a method for targeting the delivery of a chemotherapeutic entity, radionuclide, or detection entity to cells expressing TRBC in a subject.

[0176] The term "antibody conjugate of the present invention" is described in detail in relation to the aforementioned aspects of the present invention, and its features and embodiments are equally applicable to these aspects of the present invention.

[0177] 5. Personalized medical approaches Since T-cell malignancies are clonal, all malignant cells express either TRBC1 or TRBC2. We have previously demonstrated that immunotherapy targeting either TRBC1 or TRBC2 provides the ability to treat T-cell lymphoma while potentially providing an acceptable toxicity profile (Maciocia et al., 2017, Nat Med 23:1416-23; International Publication No. 2015 / 132598). The present invention also provides a method for identifying subjects having T-cell lymphoma or leukemia suitable for treatment with the antibody conjugate of the present invention, comprising determining the proportion of TRBC1-positive or TRBC2-positive T cells in a T-cell-containing sample from the subject.

[0178] Therefore, in another aspect, the present invention relates to a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Selecting an antibody conjugate for use according to the present invention based on the TRBC1 or TRBC2 expression of the malignant T cells; The present invention relates to a method including, hereinafter referred to as "the first method of personalized medicine of the present invention."

[0179] The terms “antibody conjugate of the present invention” and “substance” are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0180] In the first method of personalized medicine according to the present invention, if the proportion of TRBC1-positive T cells in the sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater, the subject is suitable to receive therapy based on a conjugated antibody for use according to the present invention that is specific to TRBC1. Similarly, if the proportion of TRBC2-positive T cells in the sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater, the subject is suitable to receive the treatment with the antibody conjugate of the present invention that is specific to TRBC2.

[0181] In another aspect, the present invention relates to a method for selecting subjects suffering from T-cell lymphoma or leukemia to receive a therapy comprising an antibody conjugate for use according to the present invention, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Selecting subjects to receive antibody conjugate-based therapy for use according to the present invention, based on the TRBC1 or TRBC2 expression of the malignant T cells; The present invention relates to a method including, hereinafter referred to as "the second method of personalized medicine of the present invention."

[0182] The terms “antibody conjugate of the present invention” and “substance” are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0183] In a second method of personalized medicine according to the present invention, if the proportion of TRBC1-positive T cells in the sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater, the subject is suitable to receive therapy based on a conjugated antibody for use according to the present invention that is specific to TRBC1. Similarly, if the proportion of TRBC2-positive T cells in the sample is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or greater, the subject is suitable to receive the treatment with the antibody conjugate according to the present invention that is specific to TRBC2.

[0184] In the first and second methods of personalized medicine of the present invention, T cell-containing samples, such as biological samples, are obtained from the subject being tested. As used herein, the terms “sample” or “biological sample” include sections of different types of biological fluids or tissues of an affected organ containing T cells. Exemplary, non-limiting examples of samples useful in the diagnostic methods of the present invention include various types of biological fluids containing T cells, such as blood, lymph, and cerebrospinal fluid. These biological fluid samples can be obtained by any conventional method known to those skilled in the art. Alternatively, the samples may also be sections of tissue from an affected organ, such as a lymph node, spleen, tonsil, or thymus, obtained from frozen sections taken for histological purposes, in addition to any conventional method, such as biopsy or surgical excision.

[0185] The sample may be a blood sample or may be derived from a blood sample.

[0186] A first step (i) to determine whether malignant T cells in a sample isolated from a subject express TRBC1 or TRBC2 may be carried out using an anti-TRBC1 and / or anti-TRBC2 antibody, such as those described in the first aspect of the present invention. This first step may use an anti-CD3 antibody, such as OKT3, to determine the total number of T cells.

[0187] There are several immunological methods available for determining whether malignant T cells express conventional TRBC1 or TRBC2. Non-limiting examples include immunohistochemistry and flow cytometry.

[0188] T cell lymphoma or leukemia can be selected from peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T cell lymphoma (EATL), hepatosplenic T cell lymphoma (HSTL), extranodal NK / T cell lymphoma, nasal type, cutaneous T cell lymphoma, primary cutaneous ALCL, T cell prolymphocytic leukemia, and T cell acute lymphoblastic leukemia.

[0189] 6. Other aspects of the invention 6.1. TRBC1-specific antibody In a further aspect, the invention provides an antibody that specifically binds to TRBC1, hereinafter "the TRBC1-specific antibody of the invention", comprising a combination of the following mutations in the VH domain as 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: - G106A in the VH domain; - Y102M in the VH domain; - G26P and T28K in the VH domain; and - G31S in the VH domain The antibody may comprise one of the above.

[0190] The antibody for use in the antibody conjugate of the invention that is specific for TRBC2 may consist of 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, together with the following mutations: - G106A in the VH domain; - Y102M in the VH domain; - G26P and T28K in the VH domain; and - G31S in the VH domain The antibody may consist of 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, together with the above mutations.

[0191] The term "antibody" is described in detail in the context of the first aspect of the present invention, and its features and aspects are equally applicable to this aspect of the present invention.

[0192] The antibody of the present invention can be an antibody fragment that maintains the ability to specifically bind to TRBC1. The antibody fragment can be an antigen-binding domain such as scFv or Fab.

[0193] 6.2. Antibodies specific for TRBC2 In a further aspect, the present invention provides an antibody that specifically binds to TRBC1 and comprises one of the following mutations in the VH domain as 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: - T28R, Y32F and A100N in the VH domain Hereinafter referred to as "the TRBC2-specific antibody of the present invention".

[0194] The antibody for use in an antibody conjugate of the present invention that is specific for TRBC2 can consist of 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, together with the following mutations: - T28R, Y32F and A100N in the VH domain Hereinafter referred to as "the TRBC2-specific antibody of the present invention".

[0195] The term "antibody" is described in detail in the context of the first aspect of the present invention, and its features and aspects are equally applicable to this aspect of the present invention.

[0196] The antibody of the present invention can be an antibody fragment that maintains the ability to specifically bind to TRBC1. The antibody fragment can be an antigen-binding domain such as scFv or Fab.

[0197] 6.2. Chimeric antigen receptor In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the TRBC1-specific antibody or the TRBC2-specific antibody of the present invention, a spacer, a transmembrane domain, and an endodomain, hereinafter referred to as "the CAR of the present invention."

[0198] The terms "TRBC1-specific antibody of the present invention" and "TRBC2-specific antibody of the present invention" are described in detail in the preceding section, and their features and embodiments apply equally to this section of the present invention.

[0199] As used herein, the terms “chimeric antigen receptor,” “CAR,” “chimeric T cell receptor,” “artificial T cell receptor,” or “chimeric immunoreceptor” refer to chimeric type I transmembrane proteins that connect an extracellular antigen-recognition domain (conjugate) to an intracellular signaling domain (endodomain). The conjugate is typically a single-stranded variable fragment (scFv) derived from a monoclonal antibody (mAb), but 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 for proper orientation of the conjugate. A common spacer domain used is the Fc of IgG1. A more compact spacer may be sufficient depending on the antigen, for example, a stalk from CD8α, or even just the IgG1 hinge. The transmembrane domain fixes the protein to the cell membrane and connects the spacer to the endodomain.

[0200] Early CAR designs had endodomains derived from either the γ chain of FcεR1 or the intracellular portion of CD3ζ. As a result, these first-generation receptors transmitted immunological signal 1, which was sufficient to trigger T cell killing of congeneral target cells, but could not fully activate T cells to proliferate and survive. To overcome this limitation, compound endodomains have been constructed; i.e., the fusion of the intracellular portion of a T cell costimulatory molecule to the intracellular portion of CD3ζ results in second-generation receptors that can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This delivers the most potent costimulatory signal, i.e., immunological signal 2, which triggers T cell proliferation. Several receptors containing TNF receptor family endodomains, such as the closely related OX40 and 4-1BB, which transmit survival signals, have also been described. Even more potent third-generation CARs with endodomains capable of transmitting activation, proliferation, and survival signals are now being described.

[0201] When a CAR binds to a target antigen, this results in the transmission of an activation signal to T cells expressing the target antigen. Thus, the CAR directs the specificity and cytotoxicity of T cells towards tumor cells expressing the targeted antigen.

[0202] Therefore, CARs typically include (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) a signaling domain, or an intracellular domain that associates with a signaling domain (see Figure 4). CAR is a general structure: It may possess an antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signal transduction domain (endodomain).

[0203] The CAR of the present invention may include a signal peptide such that when the CAR is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface and is expressed on the cell surface.

[0204] The core of the signal peptide may contain a long stretch of hydrophobic amino acids that tend to form a single α helix. The signal peptide may begin with a short stretch of positively charged amino acids that help to reinforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, there is typically a stretch of amino acids that are recognized and cleaved by signal peptidase. Signal peptidase may cleave during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by specific proteases.

[0205] The signal peptide may be present at the amino terminus of the molecule.

[0206] The signal peptide may include variants thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions), provided that the signal peptide still functions to cause cell surface expression of the protein, such as SEQ ID NO: 3-5. SEQ ID NO: 3: MGTSLLCWMALCLLGADHADG

[0207] The signal peptide of SEQ ID NO: 3 is compact and highly efficient. It is predicted to give about 95% cleavage behind the terminal glycine, providing efficient removal by signal peptidase. SEQ ID NO: 4: MSLPVTALLLPLALLLHAARP

[0208] The signal peptide of SEQ ID NO: 4 is derived from IgG1. SEQ ID NO: 5: MAVPTQVLGLLLLWLTDARC

[0209] The signal peptide of SEQ ID NO: 5 is derived from CD8.

[0210] 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.

[0211] In the CAR of the present invention, the spacer sequence may include, for example, an IgG1Fc region, an IgG1 hinge, or a human CD8 stalk or a mouse CD8 stalk. Alternatively, the spacer may include an alternative linker sequence having similar length and / or domain spacing characteristics to the IgG1Fc region, IgG1 hinge, or CD8 stalk. 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 International Publication No. 2016 / 151315.

[0212] The CAR of the present invention may include sequences selected from the sequences shown as SEQ ID NOs: 6 to 10 or variants thereof having at least 80% sequence identity. [ka] [ka]

[0213] It is possible to cleave the COMP coiled-coil domain at the N-terminus while retaining surface expression. Therefore, the coiled-coil COMP spacer may contain, or consist of, a cleaved form of SEQ ID NO: 18 cleaved at the N-terminus. The cleaved COMP may contain the five C-terminal amino acids of SEQ ID NO: 19, i.e., sequence CDACG (SEQ ID NO: 20). The cleaved COMP may contain 5 to 44 amino acids, for example, at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. The cleaved COMP may correspond to the C-terminus of SEQ ID NO: 19. For example, a cleaved COMP containing 20 amino acids may contain the sequence QQVREITFLKNTVMECDACG (SEQ ID NO: 21). The cleaved COMP may retain cysteine ​​residues involved in multimerization. The cleaved COMP may retain the ability to form multimers.

[0214] A transmembrane domain is a sequence of CARs that spans across the membrane.

[0215] A transmembrane domain can be any protein structure that is thermodynamically stable within a membrane. Typically, this is an alpha-helix composed of several hydrophobic residues. A transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the present invention. The presence and full length of a 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 a protein's transmembrane domain is a relatively simple structure, i.e., a polypeptide sequence expected to form a hydrophobic alpha-helix of sufficient length to span the membrane, artificially designed TM domains can also be used (U.S. Patent 7052906 B1 describes a synthetic transmembrane component).

[0216] The transmembrane domain may be derived from CD28, CD8a, or TYRP-1, which provides excellent receptor stability.

[0217] In one embodiment, the transmembrane domain is derived from CD8a. Sequence ID 11: CD8a transmembrane domain IYIWAPLAGTCGVLLLSLVIT

[0218] In another embodiment, the transmembrane domain is derived from TYRP-1. Sequence ID 12: TYRP-1 transmembrane domain IIAIAVVGALLLVALIFGTASYLI

[0219] The endodomain is the signaling portion of a CAR. After antigen recognition, the receptors cluster, and native CD45 and CD148 are detached from the synapse, transmitting signals to the cell. The most commonly used endodomain component is the CD3ζ endodomain component containing three ITAMs. This transmits an activation signal to T cells after antigen binding. CD3ζ may not provide a fully capable activation signal, and further co-stimulatory signaling may be required. Examples of co-stimulatory domains include endodomains derived from CD28, OX40, 4-1BB, CD27, and ICOS, which can be used with CD3ζ to transmit proliferation / survival signals.

[0220] In one embodiment, at least one co-stimulatory endodomain is used in conjunction with CD3ζ. In a particular embodiment, the co-stimulatory endodomain is selected from the group consisting of endodomains derived from CD28, OX40, 4-1BB, CD27, and ICOS.

[0221] In another embodiment, at least two co-stimulatory endodomains are used in conjunction with CD3ζ. In a particular embodiment, the two co-stimulatory endodomains are selected from the group consisting of endodomains derived from CD28, OX40, 4-1BB, CD27, and ICOS, in any combination and order. Particularly suitable combinations include endodomains derived from CD28 and CD3ζ, endodomains of OX40 and CD3ζ, endodomains of 4-1BB and CD3ζ, endodomains derived from CD28, OX40, and CD3ζ, and endodomains derived from CD28, 4-1BB, and CD3ζ.

[0222] The transmembrane and intracellular T cell signaling domains (endodomains) of CARs having an activated endodomain may include the sequences shown as Sequence IDs 13-18, or variants thereof having at least 80% sequence identity. [ka] [ka]

[0223] Insofar as the sequence provides an effective transmembrane domain and an effective intracellular T cell signaling domain, the mutant sequence may have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NOs. 13-18.

[0224] A wide variety of molecules have been developed based on the fundamental concept of having two antibody-like binding domains.

[0225] Bispecific T cell inducing molecules are a class of bispecific antibody molecules developed primarily for use as anticancer agents. Bispecific T cell inducing molecules direct the host immune system, more specifically the cytotoxic activity of T cells, against target cells such as cancer cells. In these molecules, one binding domain binds to T cells via the CD3 receptor, and the other binds to target cells such as tumor cells (via tumor-specific molecules). Because bispecific molecules bind to both target cells and T cells, they bring target cells closer to T cells, allowing T cells to exert their effects, such as cytotoxic effects against cancer cells. The formation of a T cell:bispecific antibody:cancer cell complex induces signaling in T cells, leading to, for example, the release of cytotoxic mediators. Ideally, the active agent induces the desired signaling in the presence of target cells, resulting in selective cell death.

[0226] Bispecific T-cell inducing molecules have been developed in several different forms, but one of the most common is a fusion consisting of two single-stranded variable fragments (scFv) of different antibodies arranged in series. These are sometimes known as BiTEs (Bi-specific T-cell Engagers).

[0227] 6.3. Bispecific T cell inducers The present invention also aims to provide a bispecific molecule that can selectively recognize TRBC1, induce T cells, and activate them. For example, the molecule could be BiTE.

[0228] Therefore, in another aspect, the present invention provides a bispecific T cell inducer (BiTE) comprising the TRBC1-specific antibody or the TRBC2-specific antibody of the present invention and a T cell activation domain, hereinafter referred to as "the BiTE of the present invention".

[0229] The terms "TRBC1-specific antibody of the present invention" and "TRBC2-specific antibody of the present invention" are described in detail in the preceding section, and their features and embodiments apply equally to this section of the present invention.

[0230] 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 purposes of the present invention are well known in the art and include, but are not limited to, scFvs derived from OKT3.

[0231] The bispecific molecule may contain a signal peptide that assists in its production. The signal peptide may cause the host cell to secrete the bispecific molecule so that it can be harvested from the host cell supernatant.

[0232] The signal peptide may be located at the amino terminus of the molecule. The bispecific molecule may have the general formula: signal peptide - mutant antigen-binding domain of the present invention - T cell activation domain.

[0233] The bispecific molecule may include a spacer sequence to connect the mutant antigen-binding domain and the T cell activation domain of the present invention and to spatially separate the two domains.

[0234] The spacer sequence may include, for example, an IgG1 hinge or a CD8 stalk. Alternatively, the linker may include an alternative linker sequence having similar length and / or domain spacing characteristics to the IgG1 hinge or CD8 stalk.

[0235] 6.4. Nucleic acids: In another aspect, the present invention also provides nucleic acid sequences encoding the TRBC1-specific antibody or the TRBC2-specific antibody of the present invention.

[0236] In another aspect, the present invention also provides nucleic acid sequences encoding the CAR of the present invention.

[0237] In another aspect, the present invention also provides nucleic acid sequences encoding the BiTE of the present invention.

[0238] The terms “TRBC1-specific antibody of the present invention,” “TRBC2-specific antibody of the present invention,” “CAR of the present invention,” and “BiTE of the present invention” are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0239] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with each other.

[0240] As a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that numerous different polynucleotides and nucleic acids may encode the same polypeptide. Furthermore, to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed, it should be understood that, using routine techniques, nucleotide substitutions can be made that do not affect the polypeptide sequence encoded by the polynucleotides described herein.

[0241] 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.

[0242] The nucleic acids according to the present invention may include DNA or RNA. The nucleic acids according to the present invention may be single-stranded or double-stranded. The nucleic acids according to the present invention may also be polynucleotides, which may include synthetic nucleotides or modified nucleotides. Numerous different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the use described herein, it should be understood that polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifetime of the polynucleotide of interest.

[0243] The terms “mutant,” “homologous,” or “derivative” in reference to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acids to or from the sequence.

[0244] 6.5. Vectors The present invention also provides a vector or a kit of vectors comprising one or more nucleic acids encoding the TRBC1-specific antibody of the present invention, the TRBC2-specific antibody of the present invention, the CAR of the present invention, or the BiTE of the present invention. Such vectors can be used to introduce nucleic acid sequences into host cells so that the host cells express the TRBC1-specific antibody of the present invention, or the TRBC2-specific antibody of the present invention, or the CAR of the present invention, or the BiTE of the present invention.

[0245] The terms “TRBC1-specific antibody of the present invention,” “TRBC2-specific antibody of the present invention,” “CAR of the present invention,” and “BiTE of the present invention,” as well as the nucleic acids that encode them, are described in detail in connection with the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0246] The vector may be, for example, a retroviral vector or lentiviral vector, or a transposon-based vector or a plasmid or viral vector such as synthetic mRNA.

[0247] The vector may be capable of transfecting or introducing cytolytic immune cells such as T cells or NK cells.

[0248] 6.6.Cells Another aspect of the present invention relates to cells containing the CAR of the present invention.

[0249] The cells may contain the nucleic acid or vector of the present invention.

[0250] 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 aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0251] The cells may be cytolytic immune cells such as T cells or NK cells.

[0252] T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cellular immunity. T cells, or T lymphocytes, 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. Various types of T cells exist, as summarized below.

[0253] 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 presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0254] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into an anergic state, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0255] 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 their congener antigens, memory T cells rapidly expand into a large number of effector T cells, thus providing the immune system with a "memory" of past infections. Memory T cells include 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+. Typically, memory T cells express the cell surface protein CD45RO.

[0256] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their main roles are to halt T cell-mediated immunity toward the termination of the immune response and to suppress autoreactive T cells that have escaped negative selection in the thymus.

[0257] Two major classes of CD4+ Treg cells are described: naturally occurring Treg cells and adaptive Treg cells.

[0258] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) originate in the thymus and are involved in the interaction between developing T cells and both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells 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 disrupt the development of regulatory T cells, leading to IPEX, a fatal autoimmune disease.

[0259] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0260] The cells may be natural killer cells (or NK cells). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0261] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute a third type of cell differentiated from common lymphoid progenitor cells that produce B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering circulation.

[0262] The cells of the present invention may be any of the above cell types. In one embodiment, the cells of the present invention are T cells. In another embodiment, the cells of the present invention are NK cells.

[0263] Cells according to this aspect of the present invention can be produced ex vivo from the patient's own peripheral blood (first party), or from donor peripheral blood (second party), or peripheral blood from an unrelated donor (third party), in the context of hematopoietic stem cell transplantation.

[0264] Alternatively, cells according to this aspect of the present invention may be induced from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into cytolytic cells. Or, immortalized cytolytic cell lines such as T or NK cells that retain their lytic function and can act as therapeutic agents may be used.

[0265] In all these embodiments, CAR-expressing cells are produced by introducing DNA or RNA encoding a chimeric polypeptide by one of many means, including transduction by a viral vector and transduction by DNA or RNA.

[0266] The cells of the present invention may be ex vivo cells derived from a subject. The cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. Cells, particularly cytolytic cells such as T cells or NK cells, may be activated and / or proliferated by treatment with, for example, an anti-CD3 monoclonal antibody before being transduced with a nucleic acid encoding the molecule that provides the CAR of the present invention.

[0267] The cells of the present invention can be produced by a method comprising the step of transducing or transtransferring cells with a vector of the present invention containing a nucleic acid sequence encoding a CAR.

[0268] The method for producing cells of the present invention may further include a step of isolating cells from a cell-containing sample derived from the target or from other sources listed above, prior to the transduction or transtransfer step. If the cells are cytolytic cells, the sample is a cytolytic cell-containing sample derived from the target.

[0269] As used in the context of this invention, the terms "subject" or "individual" refer to a member of a mammalian species, preferably a human being of any age or race, male or female.

[0270] Subsequently, the cells of the present invention can be purified, for example, by selection based on the expression of the antigen-binding domain of the CAR.

[0271] 6.7. Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition containing the TRBC1-specific antibody of the present invention, or the TRBC2-specific antibody of the present invention, or one or more cells of the present invention, or the BiTE of the present invention.

[0272] The terms “TRBC1-specific antibody of the present invention,” “TRBC2-specific antibody of the present invention,” “CAR of the present invention,” and “BiTE of the present invention” are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0273] The description and embodiments of the pharmaceutical compositions of the foregoing aspects of the present invention apply equally to these aspects of the present invention. Those skilled in the art will immediately know what modifications may be necessary to adapt the pharmaceutical compositions of the foregoing aspects of the present invention to these aspects.

[0274] 6.8. Treatment Methods In another aspect, the present invention provides a TRBC1-specific antibody, or a TRBC2-specific antibody, or one or more cells, or BiTE, for use in medical applications.

[0275] In another aspect, the present invention provides a method for treating T-cell lymphoma or leukemia in a subject, comprising the step of administering the TRBC1-specific antibody of the present invention, or the TRBC2-specific antibody of the present invention, or one or more cells of the present invention, or the BiTE of the present invention, to a subject in need thereof. The administration step may be in the form of the above-described pharmaceutical composition.

[0276] This aspect of the present invention may, as an alternative, be formulated as a TRBC1-specific antibody of the present invention, or a TRBC2-specific antibody of the present invention, or one or more cells of the present invention, or as BiTE of the present invention, for use in the treatment of T-cell lymphoma or leukemia.

[0277] This aspect of the present invention may, as an alternative, be formulated as the use of the TRBC1-specific antibody of the present invention, or the TRBC2-specific antibody of the present invention, or one or more cells of the present invention, or the BiTE of the present invention in the manufacture of a pharmaceutical product for treating T-cell lymphoma or leukemia.

[0278] The terms “TRBC1-specific antibody of the present invention,” “TRBC2-specific antibody of the present invention,” “CAR of the present invention,” and “BiTE of the present invention” are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0279] The method for treating T-cell lymphoma and / or leukemia relates to the therapeutic use of the antibody conjugate of the present invention, which can be administered to subjects having a pre-existing T-cell lymphoma and / or leukemia, in order 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.

[0280] The description and embodiments of the method for treating the aforementioned aspects of the present invention apply equally to these aspects of the present invention. Those skilled in the art will immediately know what modifications may be necessary to adapt the method for treating the aforementioned aspects of the present invention to these aspects.

[0281] 6.9. Diagnostic Agents TRBC1 + vs TRBC2 + The proportion of T cells derived from healthy donors is 35% versus 65%, meaning that the median percentage of total T cells expressing TRBC1 is 35% (range, 25-47%), as previously determined (Maciocia et al., 2017, Nat Med, 23:1416-23). ​​Since T-cell lymphoma or leukemia is a clonal cancer (Maciocia et al., 2017; see above), the deregulated proliferation of malignant T cells characteristic of T-cell lymphoma or leukemia is significantly distorted by TRBC1 + or TRBC2 + T cells (i.e., TRBC2) - or TRBC1 - This results in a proportion of T cells. Therefore, by specifically binding to TRBC1 and thus being able to distinguish between TRBC1 and TRBC2, the antibody of the present invention constitutes a useful active substance for the diagnosis of T-cell lymphoma or leukemia.

[0282] Therefore, in another aspect, the present invention provides a diagnostic agent comprising the TRBC1-specific antibody or the TRBC2-specific antibody of the present invention, hereinafter referred to as "the diagnostic agent of the present invention."

[0283] The terms "TRBC1-specific antibody of the present invention" and "TRBC2-specific antibody of the present invention" are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0284] The TRBC1-specific antibody or TRBC2-specific antibody of the present invention to be used in these assays may be labeled or unlabeled. As used herein, the terms “detectable label” or “labeling agent” refer to a molecular label that enables the detection, localization, and / or identification of the molecule to which the molecular label is bound, for example, by appropriate procedures and instruments for detection by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Suitable labeling agents for labeling antibodies include radionuclides, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and derivatives. As those skilled in the art will understand, unlabeled mutant antigen-binding domains and antibodies must be detected with additional reagents, such as labeled secondary antibodies. This is particularly useful to increase the sensitivity of the detection method, as it allows the signal to be amplified. A wide range of conventional assays 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, and 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 TRBC1 using the mutant antigen-binding domain or antibody of the present invention include affinity chromatography techniques or ligand-binding assays.

[0285] Diagnostic agents may be used to diagnose T-cell lymphoma or leukemia.

[0286] T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); anaplastic large cell lymphoma (ALCL); enteropathy-associated T-cell lymphoma (EATL); hepatosplenic T-cell lymphoma (HSTL); extranodal NK / T-cell lymphoma nasal type; cutaneous T-cell lymphoma; primary cutaneous ALCL; T-cell prolymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0287] 6.10. Diagnostic Methods In another 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 TRBC1-specific antibody or a TRBC2-specific antibody of the present invention.

[0288] The terms "TRBC1-specific antibody of the present invention," "TRBC2-specific antibody of the present invention," "subject," and "sample" are described in detail in relation to the aforementioned aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0289] The diagnostic method of the present invention may further include a step of determining the total number of TRBC1-positive T cells in a sample. The diagnostic method of the present invention may further include a step of determining the total number of TRBC2-positive T cells in a sample. The diagnostic method of the present invention may further include a step of determining the total number of TRBC1-positive T cells and TRBC2-positive T cells in a sample.

[0290] The diagnostic method of the present invention may further include a step of determining the total number of T cells in a sample. This step may involve using an anti-CD3 antibody such as OKT3 to determine the total number of T cells.

[0291] The diagnostic method of the present invention may further include the step of determining the percentage of TRBC1-positive T cells in a sample.

[0292] According to the first step of the diagnostic method of the present invention, the TRBC1-specific antibody of the present invention is brought into contact with a sample from a subject under study under appropriate conditions known to those skilled in the art.

[0293] The diagnostic method of the present invention may further include the step of determining the percentage of TRBC2-positive T cells in a sample.

[0294] According to the first step of the diagnostic method of the present invention, the TRBC2-specific antibody of the present invention is brought into contact with a sample from a subject under study under appropriate conditions known to those skilled in the art.

[0295] Those skilled in the art can use a number of conventional methods for detecting TRBC1 or TRBC2 in a sample that are suitable for carrying out the second step of the diagnostic method of the present invention. Immunological methods are particularly useful. Therefore, the use of the diagnostic agent of the present invention may be particularly useful for carrying out the diagnostic method according to the present invention. The features and specific embodiments of the diagnostic agent of the present invention have already been defined and apply equally to the diagnostic method of the present invention.

[0296] In the diagnostic method of the present invention, a percentage of TRBC1-positive T cells in a sample of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more may indicate the presence of T-cell lymphoma or leukemia.

[0297] As those skilled in the art will understand, 100% accuracy is preferable, but predictions do not need to be accurate for 100% of the subjects being diagnosed or evaluated. However, this term requires that a statistically significant portion of the subjects can be identified as having an increased probability of having a given outcome. Whether the data obtained from the subjects are statistically significant can be determined without difficulty by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, p-values, and cross-validated classification rates. 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 less.

[0298] Furthermore, given their ability to specifically bind TRBC1-positive T cells and TRBC2-positive T cells, respectively, the TRBC1-specific antibody and the TRBC2-specific antibody of the present invention can also be used for in vivo diagnosis of T-cell lymphoma or leukemia. For example, the TRBC1-specific antibody and the TRBC2-specific antibody of the present invention can be used in medical imaging, i.e., a series of techniques and processes used to create images of the body (or its parts and functions), such as the human body, for clinical purposes such as medical procedures aimed at revealing, diagnosing, or testing for disease.

[0299] For this purpose, the mutant antigen-binding domains or antibodies of the present invention are labeled by appropriate methods known in the art and provided as active agents for imaging diagnostics such as radioimmunodiagnosis, positron emission tomography (PET), and endoscopic immunofluorescence by, for example, conjugation and / or loading with appropriate molecules, such as radioisotopes or fluorescent dyes. The mutant antigen-binding domains and antibodies of the present invention may be conjugated to gamma-ray radioisotopes and used in radioimmunoscintigraphy using a gamma camera or single-photon emission computed tomography. The mutant antigen-binding domains and antibodies of the present invention may be conjugated to positron emitters and used in PET. The mutant antigen-binding domains and antibodies of the present invention may be conjugated to fluorescent dyes such as Cy3, Cy2, Cy5, or FITC and used in endoscopic immunofluorescence. The mutant antigen-binding domains and antibodies of the present invention, modified as described, are administered to an individual in an appropriate dose by any appropriate route, for example, intravenously, and the location of TRBC1-positive T cells is detected, determined, or measured by processes known in the art. The methods and techniques used herein, including those related to diagnostic imaging, are known to those skilled in the art, and suitable dosage formulations can also be provided to those skilled in the art.

[0300] The T-cell lymphomas or leukemias to be diagnosed may be selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); anaplastic large cell lymphoma (ALCL); enteropathy-associated T-cell lymphoma (EATL); hepatosplenic T-cell lymphoma (HSTL); extranodal NK / T-cell lymphoma nasal type; cutaneous T-cell lymphoma; primary cutaneous ALCL; T-cell prolymphocytic leukemia; and T-cell acute lymphoblastic leukemia.

[0301] The sample may be a blood sample or may be derived from a blood sample.

[0302] 6.11. Personalized medical practices In another aspect, the present invention provides a method for identifying subjects having T-cell lymphoma or leukemia suitable for treatment with the cells of the present invention, a TRBC1-specific antibody, a TRBC2-specific antibody, or BiTE, the method comprising determining the proportion of TRBC1-positive T cells and / or TRBC2-positive T cells in a sample containing T cells from said subjects.

[0303] Therefore, in another aspect, the present invention relates to a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Selecting cells, a TRBC1-specific antibody, a TRBC2-specific antibody, or a BiTE for use according to the present invention based on the TRBC1 or TRBC2 expression of the malignant T cells; The present invention relates to a method including, hereinafter referred to as "the first method of personalized medicine of the present invention."

[0304] In another aspect, the present invention relates to a method for selecting subjects suffering from T-cell lymphoma or leukemia to receive a therapy comprising cells for use according to the present invention, a TRBC1-specific antibody, a TRBC2-specific antibody, or BiTE, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Selecting subjects to receive therapy based on the TRBC1 or TRBC2 expression of malignant T cells according to the present invention, cells for use according to the present invention, TRBC1-specific antibodies, TRBC2-specific antibodies, or BiTE-based therapy; The present invention relates to a method including, hereinafter referred to as "the second method of personalized medicine of the present invention."

[0305] The terms “cells of the present invention,” “TRBC1-specific antibody of the present invention,” “TRBC2-specific antibody of the present invention,” “BiTE of the present invention,” “subject,” and “sample containing T cells” are described in detail in relation to the foregoing aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0306] The description and embodiments of the personalized medical methods of the aforementioned aspects of the present invention apply equally to these aspects of the present invention. Those skilled in the art will immediately know what modifications may be necessary to adapt the personalized medical methods of the aforementioned aspects of the present invention to these aspects. [Examples]

[0307] Examples [Example 1] Example 1: Internal transfer of hJOVI-1 and KFN antibodies The cell uptake of the anti-TRBC1 antibody hJOVI-1 and the anti-TRBC2 antibody KFN was tested in HPB-ALL cells manipulated to express only TRBC1, only TRBC2, or in which TRBCs were knocked out (KO). For this purpose, hJOVI-1 and KFN were conjugated in IgG form using Zenon pHrodo iFL red (Thermo Scientific), a pH-sensitive fluorophore with a 560 / 585 nm spectrum, according to the manufacturer's recommendations. An unrelated anti-HEL antibody was also conjugated for use as a control. The conjugated antibodies were incubated with HPB-ALL TRBC1, HPB-ALL TRBC2, and HPB-ALL KO cells at 37°C and 5% CO2 for 24 hours. 5 × 10⁶ cells were incubated in 100 μl of RPMI 10% FBS. 4Test antibodies hJOVI-1, KFN, and anti-HEL were applied at 5 μg / ml to 96-well plates containing cells / well. After 24 hours, cells were washed, harvested, stained with 1:100 LIVE / DEAD™ Fixed Violet Dead Cell Stain (Thermofisher) for 10 minutes, and then analyzed by flow cytometry using a Fortessa flow cytometer (BD).

[0308] The results shown in Figure 4 demonstrate that both the anti-TRBC antibodies hJOVI-1 and KFN successfully intermigrate into HPB-ALL cells. However, the KFN antibody, which binds to the same epitope and target, albeit with lower affinity, showed improved intermigration than the higher-affinity hJOVI-1. Since hJOVI-1 intermigrated only into HPB-ALL TRBC1 cells and KFN intermigrated only into HPB-ALL TRBC2 cells, the specificity of hJOVI-1 and KFN was maintained as antibody conjugates.

[0309] [Example 2] Example 2: Affinity and binding kinetics of TRBC antibodies Surface plasmon resonance (SPR) was performed on several antibody mutants of hJOVI-1 to confirm affinity, association, and dissociation rates. The sequence details of these mutants Mut1-Mut15 used in the Examples section of this patent application are shown in Tables 1 and 2. These anti-TRBC antibody mutants are specific to either TRBC1 or TRBC2.

[0310] SPR experiments were performed using a Biacore T200 instrument with HBSP1 (GE Healthcare BioSciences) as the running and dilution buffer. BIAevaluation software version 2.0 (GE Healthcare) was used for data processing. For determining binding kinetics, mouse anti-human IgG (GE Healthcare) was covalently bound to a CM5 Sensor Chip (GE Healthcare). Anti-TRBC1 or anti-TRBC2 antibody was captured on a flow cell, and various concentrations of interaction partner proteins (TRBC1 or TRBC2) were injected onto the flow cell at a flow rate of 30 ml / min at a temperature of 25°C. Double reference subtraction was performed using buffer only. Kinetic rate constants were obtained by curve fitting following a 1:1 Langmuir binding model.

[0311] The affinity and kinetic rate constants obtained by SPR are shown in Figure 5 and Table 1.

[0312] Various mutants with different binding properties to TRBC1 were selected, and their kinetic profiles for TRBC1 were compared (Figure 6). Several binders with very similar association rates but different dissociation rates were identified and further evaluated.

[0313] [Example 3] Example 3: Internal transfer of anti-TRBC1 antibody The internal migration of the anti-TRBC1 antibody selected in Example 2 (Figure 6) was evaluated in HPB-ALL cells (TRBC1, TRBC2, and KO). For this purpose, the anti-TRBC1 antibody was conjugated in IgG form according to the manufacturer's recommendations using the pH-sensitive fluorophore Zenon pHrodo iFL Green reagent (Thermo Scientific) with a 509 / 533 nm spectrum. An unrelated anti-HEL antibody was also conjugated for use as a control. The conjugated antibodies were incubated with HPB-ALL TRBC1, HPB-ALL TRBC2, and HPB-ALL KO cells at 37°C and 5% CO2 for 9 hours. 5 × 10⁶ cells were incubated in 100 μl of RPMI 10% FBS. 4 The test antibody was applied at a concentration of 5 μg / ml to 96-well plates containing cells / well. After 9 hours, the cells were washed, harvested, stained with the fixable survival dye eFluor® 780 (eBioscience) at a ratio of 1:1000 for 10 minutes, and then analyzed by flow cytometry using a Fortessa flow cytometer (BD).

[0314] After 9 hours, cells were washed with PBS, harvested at 400g for 5 minutes, stained with a 1:1000 fixable survival dye eFluor® 780 (eBioscience®, Thermofisher) for 10 minutes, and then analyzed by flow cytometry using a Fortessa flow cytometer (BD).

[0315] The results demonstrated that all anti-TRBC1 antibodies were adequately internalized (Figure 7). Analysis of the internalization ability of these mutants showed that antibodies with reduced affinity and increased dissociation rate were more readily internalized. However, internalization was impaired at a specific affinity (Mut14), which likely represents a lack of target antigen binding. This is surprising, as previous reports had claimed that high affinity, and therefore slow dissociation rate, is required to achieve optimal cellular uptake.

[0316] [Example 4] Example 4: Selection of anti-TRBC1 and anti-TRBC2 antibodies for optimal ADCs Based on the findings of Example 3, namely that improved internal translocation occurs at lower affinity, the anti-TRBC1 antibody Mut11 was selected for further investigation because it mimics the dissociation profile of anti-TRBC2 clone KFN. Similarly, the TRBC2 antibody Mut15 was also selected because it exhibits optimal binding kinetics.

[0317] An internal migration experiment using Mut11 and Mut15 was performed as described in Example 3.

[0318] The results showed that both Mut11 and Mut15 underwent good internal migration (Figure 8). Furthermore, the results demonstrated that the internal migration achieved with these two antibodies was significantly improved compared to the internal migration of hJovi1. Mutant Mut11 showed the greatest internal migration in TRBC1 cells while maintaining antigen specificity. Similarly, KFN-mutant Mut15 showed optimal internal migration and specificity to TRBC2 cells.

[0319] [Example 5] Example 5: Fabrication of TRBC-specific ADCs To test the efficacy of TRBC1 and TRBC2 ADC molecules, Mut11 and Mut15 antibodies were conjugated to monomethyl auristatin E (MMAE). A control anti-HEL was used. Briefly, antibodies were prepared and purified to high purity. Antibody-drug conjugates were generated using the linker-loaded mc-vc-PAB-MMAE (Figure 9A). Briefly, a reducing agent was first used to release nucleophilic cysteine ​​residues from the interchain disulfide bonds of the reduced antibody. The reduced cysteine ​​was conjugated with the drug-linker complex. Complex formation conditions were optimized to identify ADCs with the desired drug-to-antibody ratio (DAR). The drug-to-antibody ratio was evaluated by hydrophobic interaction chromatography on a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 100 mm) and was evaluated at 3.1, 3.2, and 3.3 for Mut11, Mut15, and anti-HEL, respectively.

[0320] Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the antibody's ability to internally transfer (Figure 9B).

[0321] [Example 6] Example 6: Determination of the functional characteristics of TRBC-specific ADCs 5 × 10¹⁶ cells stimulated with 10 ng / ml human IL-7 5 In vitro cytotoxicity assays were performed on HPB-ALL TRBC1+, TRBC2+, and TCR KO cells / ml. ADC constructs were incubated with target cells at a concentration of 8 μg / ml at 37°C for 116 hours, followed by incubation with 10% culture volume of Alamar Blue for 4 hours. The supernatant was acquired using a plate reader (Varioscan Lux, Thermo Scientific) with a 560 / 590 nm (excitation / emission) filter setting. Viability was expressed as standardization by untreated cells (treated × 100 / untreated). IC50 was measured for each test compound with an unconjugated control compound to determine the concentration at which maximum half of the cells were inhibited.

[0322] To test the efficacy of TRBC1 and TRBC2 ADC molecules, MMAE-conjugated Mut11 and Mut15 antibodies were tested for their ability to induce cell death. The maximum half-dose inhibitory concentrations at which Mut11-MMAE and Mut15-MMAE induced cell death in TRBC1-expressing cells and TRBC2-expressing cells were 0.14 and 0.34 μg / ml, respectively (Figure 9C). These results demonstrate that anti-TRBC1 and anti-TRBC2 ADC molecules are effective in inducing internal translocation leading to cell death.

[0323] [Example 7] Example 7: Fabrication of anti-TRBC1 CAR based on hJOVI-1 The second-generation CAR construct (Figure 4) has the following combination of mutations compared to hJOVI1 (which has 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): -G106A in the VH domain; -Y102M in the VH domain; - G26P and T28K in the VH domain; and - G31S in VH domain It is generated based on the following anti-TRBC1 antibodies, including one of the following:

[0324] These CAR constructs are cloned into retroviral vectors and used to transduce activated PBMCs obtained from healthy donors.

[0325] [Example 8] Example 8: Determination of the functional characteristics of anti-TRBC1 CAR: cytokine production To evaluate the functional capacity of anti-TRBC1 mutant CAR-T cells against TRBC1, a plate-binding assay is used in which TRBC1 or TRBC2 is immobilized before the addition of CAR-T cells. After 72 hours, the culture supernatant is collected and IFN-γ production is measured by ELISA.

[0326] [Example 9] Example 9: Functional characterization of anti-TRBC1 CAR: Cytotoxic assay To determine the ability of anti-TRBC1 triple mutants to target TRBC1, 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. (Raji WT, Raji TRBC1) + Or Raji TRBC2 + hJovi-1 CAR-T cells or anti-TRBC1 triple mutant CAR-T cells are cultured with either of the cell types in a 1:1 (E:T) ratio. Target cell retrieval is measured by flow cytometry after 72 hours of culture and used to establish the cytotoxic capacity of the CAR-T cells.

[0327] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been 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 embodiments for carrying out the present invention, which will be apparent to those skilled in the art in molecular biology or related fields, are intended to fall within the scope of the following claims. The present invention provides, for example, the following items: (Item 1) An antibody conjugate that specifically binds to the constant region (TRBC) of the TCRβ chain, wherein the antibody is 0.001 seconds -1 ~0.3 seconds -1 Dissociation rate constant (k) in the range d An antibody conjugate having ) (Item 2) The aforementioned antibody, 0.002 seconds -1 ~0.1 seconds -1 k within the range d The antibody conjugate described in item 1, having the properties of the antibody conjugate described in item 1. (Item 3) The antibody conjugate according to item 1 or 2, wherein the antibody is conjugated to a chemotherapeutic entity, a radionuclide, or a detection entity. (Item 4) The antibody conjugate described in item 3, wherein the aforementioned chemotherapy entity is a tubulin inhibitor. (Item 5) The antibody conjugate described in item 4, wherein the tubulin inhibitor is an MMAE. (Item 6) The antibody conjugate according to any one of items 1 to 5, wherein the antibody conjugate has increased internal migration upon binding to target cells compared to the internal migration of 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. (Item 7) An antibody conjugate according to any one of items 1 to 6, wherein the antibody specifically binds to TRBC1. (Item 8) 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, the antibody exhibits the following mutations or combinations of mutations: - G106A in the aforementioned VH domain; -Y32F in the aforementioned VH domain; - G31S in the aforementioned VH domain; - G26P and T28K in the VH domain; or -Y102M in the VH domain An antibody conjugate as described in item 7, including one of the following. (Item 9) The antibody exhibits the following mutations 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: - G106A in the VH domain Antibody conjugates, including those listed in item 8. (Item 10) An antibody conjugate according to any one of items 1 to 6, wherein the antibody specifically binds to TRBC2. (Item 11) 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, the antibody exhibits the following mutations or combinations of mutations: -T28K, Y32F, A100N, Y102L and N103M in the VH domain, and N35R in the VL domain; -T28K, Y32F, and A100N in the VH domain; or -T28R, Y32F and A100N in the VH domain An antibody conjugate as described in item 10, including one of the following. (Item 12) 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, the antibody exhibits the following mutation combinations: -T28K, Y32F, A100N and N103L in the VH domain, and N35R in the VL domain An antibody conjugate as described in item 11, including one of the following. (Item 13) An antibody conjugate as described in any one of items 1 to 12, for use in the treatment of T-cell lymphoma or leukemia. (Item 14) An antibody conjugate for use according to item 13, comprising the step of administering the antibody conjugate to the subject so that the treatment for T-cell lymphoma or leukemia in the subject causes selective depletion of the malignant T cells, along with normal T cells that express the same TRBC as the malignant T cells, but does not cause depletion of normal T cells that express TRBCs not expressed by the malignant T cells. (Item 15) The antibody conjugate for use according to item 14, wherein the method further comprises the step of examining the constant region of the TCRβ chain (TCRB) of malignant T cells derived from the subject in order to determine whether the malignant T cells derived from the subject express TRBC1 or TRBC2. (Item 16) The aforementioned T-cell lymphoma or leukemia is selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia, and is an antibody conjugate for use as described in any of items 13-15. (Item 17) An antibody conjugate as described in any of items 3 to 12, for use in methods for targeting the delivery of chemotherapeutic agents to cells expressing TRBCs in the subject. (Item 18) A pharmaceutical composition comprising an antibody conjugate described in any of items 1 to 12, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. (Item 19) A method for selecting an appropriate therapy to treat a subject suffering from T-cell lymphoma or leukemia, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Select an antibody conjugate for use as described in any of items 13-16, based on the TRBC1 or TRBC2 expression of the malignant T cells; Methods that include... (Item 20) A method for selecting a subject suffering from T-cell lymphoma or leukemia to receive therapy containing an antibody conjugate for use as described in any of items 13-16, i) To determine whether malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2; ii) Selecting the subject to receive antibody conjugate-based therapy for use as described in any of items 13-16, based on the TRBC1 or TRBC2 expression of the malignant T cells; Methods that include...

Claims

1. An antibody conjugate that binds to TRBC1 or TRBC2, wherein the antibody is 0.001 seconds －１ ~0.3 seconds －１ Dissociation rate constant (k) in the range ｄ ) has, The antibody that binds to TRBC1, 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, exhibits the following mutations or combinations of mutations: - G106A in the VH domain; - G26P and T28K in the VH domain; or - Y102M in the aforementioned VH domain It includes one of the following: The antibody that binds to TRBC2 exhibits the following mutations or combinations of mutations 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: - T28K, Y32F, A100N, Y102L and N103M in the VH domain, and N35R in the VL domain; or - T28K, Y32F and A100N in the VH domain It includes one of the following: The antibody is conjugated with a chemotherapeutic entity, a radionuclide, or a detection entity. The aforementioned chemotherapeutic substance is a cytotoxic drug, cytotoxic agent, or biological response modifier. An antibody conjugate in which the detection entity is a fluorescent peptide, dye, or label.

2. The aforementioned antibody lasted 0.002 seconds －１ ~0.1 seconds －１ k within the range ｄ The antibody conjugate according to claim 1, having the following characteristics.

3. The antibody conjugate according to any one of claims 1 or 2, wherein the chemotherapy substance is a tubulin inhibitor, and the tubulin inhibitor is vincristine, vinblastine, vinflunin, halichondrin B, eribulin mesylate, cryptophysin, drastatin, mytansin, mytansinoid, colchicine, 2-methoxyestradiol, sulfonamide, paclitaxel, docetaxel, cyclostreptin, eryuterobin, ABI-007, ixabepylone, patupylone, BMS-310705, laurimaride, or perolcid A.

4. The antibody conjugate according to claim 3, wherein the aforementioned drastatin is MMAE.

5. The antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate has increased internal migration upon binding to target cells compared to the internal migration of 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.

6. A composition comprising an antibody conjugate according to any one of claims 1 to 5 for use in the treatment of T-cell lymphoma or leukemia.

7. The composition according to claim 6, characterized in that the treatment for T-cell lymphoma or leukemia in the subject causes selective depletion of malignant T cells, along with normal T cells expressing the same TRBCs as the malignant T cells, but does not cause depletion of normal T cells expressing TRBCs that are not expressed by the malignant T cells, by administering the composition to the subject.

8. The composition according to claim 7, wherein the method further comprises the step of examining the TCRβ chain constant region (TCRB) of malignant T cells derived from the subject in order to determine whether the malignant T cells derived from the subject express TRBC1 or TRBC2.

9. The composition according to any one of claims 6 to 8, wherein the T-cell lymphoma or leukemia is selected from peripheral T-cell lymphoma, unspecified type (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); anaplastic large cell lymphoma (ALCL); enteropathy-associated T-cell lymphoma (EATL); hepatosplenic T-cell lymphoma (HSTL); extranodal NK / T-cell lymphoma nasal type; cutaneous T-cell lymphoma; primary cutaneous ALCL; T-cell prolymphoblastic leukemia; and T-cell acute lymphoblastic leukemia.

10. A composition comprising an antibody conjugate according to any one of claims 1 to 6 for targeting the delivery of a chemotherapeutic agent to cells expressing TRBCs in a target.

11. A pharmaceutical composition comprising an antibody conjugate according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier, diluent, excipient, or auxiliary agent.

12. A method for determining the expression of TRBC1 or TRBC2 proteins in malignant T cells in a sample isolated from a subject suffering from T-cell lymphoma or leukemia, as an indicator for selecting an appropriate therapy for treating the subject, wherein the method is To determine whether the malignant T cells in the sample express TRBC1 or TRBC2. A method comprising the expression of the TRBC1 or TRBC2 protein on the malignant T cells, wherein the expression of the TRBC1 or TRBC2 protein on the malignant T cells indicates selection of the composition according to any one of claims 6 to 9.

13. A method for determining the TRBC1 or TRBC2 protein expression of malignant T cells in a sample isolated from a subject, as an indicator for selecting a subject suffering from T-cell lymphoma or leukemia to receive therapy using the composition according to any one of claims 6 to 9, wherein the method is To determine whether the malignant T cells in the sample isolated from the subject express TRBC1 or TRBC2. A method comprising the expression of the TRBC1 or TRBC2 protein on the malignant T cells, wherein the selection of the target is indicated.

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