TRBC β antibody conjugate

Abstract

To provide a TRBC β antibody conjugate.SOLUTION: The present disclosure provides an antibody conjugate that binds specifically to TCR β constant region (TRBC), wherein the antibody has a fast dissociation rate constant (kd). The present invention further provides medical uses and methods of personalised medicine that exploit the products of the invention. The present invention provides, for example, an antibody conjugate that binds specifically to the TCR β constant region (TRBC), wherein the antibody has a dissociation rate constant (kd) in the range of 0.001 s-1 to 0.3 s-1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a conjugate of an antibody that specifically binds to the TCR β chain constant region (TRBC), 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 derived from either T cells or B cells. T-cell malignancies are a clinically and biologically heterogeneous group of disorders, collectively accounting for 10–20% of non-Hodgkin's lymphomas and 20% of acute leukemias. The most commonly identified histologic subtypes are peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL). Of all acute lymphoblastic leukemias (ALL), approximately 20% are of the T-cell phenotype.

[0003] These conditions typically behave aggressively, with an estimated 5-year survival rate of only 30%, compared to, for example, B-cell malignancies. In the case of T-cell lymphomas, a high proportion of patients present with disseminated disease, unfavorable International Prognostic Index (IPI) scores, and a prevalence of extranodal disease. Chemotherapy alone is usually ineffective, with fewer than 30% of patients being 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 immunotherapeutic agents available for the treatment of T-cell malignancies. A key difficulty in developing immunotherapies for T-cell disorders is the considerable overlap in marker expression between clonal and normal T cells, and no single antigen can unambiguously identify clonal (malignant) cells.

[0005] A targeting strategy based on the mutually exclusive expression of T cell receptor beta chain constant domains 1 and 2 (TRBC1 and TRBC2) has been reported (WO 2015 / 132598; Maciocia et al., 2017, Nat Med 23:1416-23). ​​Furthermore, it has been demonstrated that CARs targeting either TRBC1 or TRBC2 may offer the ability to treat T cell lymphoma while providing an acceptable toxicity profile (Maciocia et al., 2017, Nat Med 23:1416-23; WO 2015 / 132598).

[0006] However, diseases such as human T-cell leukemia virus, type 1 (HTLV-1)-associated leukemia and lymphoma, despite their potential for treatment with TRBC1 / TRBC2-targeted drugs, may be less amenable to cell therapy because cell-based therapies may be prone to T cell-mediated friendly fire.

[0007] Thus, there is a need in the art to provide alternative targeted agents that overcome the potential drawbacks of cell-based therapies in the treatment of T-cell lymphomas and leukemias.

[0008] Antibody-drug conjugates (ADCs) offer another immunotherapy modality that can be used to target T-cell lymphoma. ADCs offer an additional advantage over cell-based therapies in that they are less susceptible to friendly fire mediated by T cells. Furthermore, ADCs may offer improved management of side effects.

[0009] The efficiency of ADC strategies is highly dependent on the internalization of cytotoxic conjugates into cancer cells. Although ADCs based on TRBC-specific antibodies have been previously described (WO 2015 / 132598), their internalization properties are unknown.

[0010] An important feature of an antibody suitable for ADC is that it specifically binds to TRBCs and has a high internalization ability. The internalization ability of an antibody depends on the properties of both the target antigen and the antibody. It is difficult to predict an antigen-binding site suitable for internalization from the molecular structure of the target, or to easily predict an antibody with a high internalization ability from the binding strength, physical properties, etc. of the antibody. Therefore, a key challenge in developing a highly potent ADC is to obtain an antibody with a high internalization ability against the target antigen.

[0011] Therefore, there is a need in the art for TRBC-specific ADCs with optimal internalization properties. [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 Summary of the Invention [Means for solving the problem]

[0014] Summary of Aspects of the Invention The present inventors investigated ADC therapeutic strategies for T-cell lymphoma and leukemia using a number of TRBC-specific antibodies with various affinities to clarify the internalization properties of TRBC-specific antibodies upon binding to T cells. The results obtained helped identify the binding affinity that confers good internalization properties to specific antibodies. Unexpectedly, the present inventors determined that a fast dissociation rate constant is important for achieving good antibody internalization. These results contrast with the commonly accepted view in the art that high-affinity antibodies, and therefore antibodies with slow dissociation rate constants, exhibit high internalization capabilities.

[0015] Thus, in a first aspect, the present invention provides an antibody conjugate that specifically binds to a TCR beta chain constant region (TRBC), wherein the antibody is -1 ~0.3 seconds -1 The dissociation rate constant (k d )

[0016] Antibodies take 0.002 seconds -1 ~0.1 seconds -1 k in the range d may have:

[0017] The antibody may be conjugated to a chemotherapeutic entity, a radionuclide or a detection entity.

[0018] The chemotherapeutic entity may be a tubulin inhibitor.

[0019] The tubulin inhibitor can be MMAE.

[0020] The antibody conjugate may have increased internalization upon binding to a target cell compared to the internalization of a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2.

[0021] The antibody can specifically bind to TRBC1.

[0022] The TRBC1-specific antibody has the following mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: - 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 may include one of:

[0023] The TRBC1-specific antibody has the following mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: G106A in the VH domain may include:

[0024] The antibody can specifically bind to TRBC2.

[0025] The TRBC2-specific antibody has the following combination of mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: - 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 may include one of:

[0026] The TRBC2-specific antibody has the following mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: - T28K, Y32F, A100N, Y102L and N103M in the VH domain and VL N35R in the VL domain may include: In a second aspect, the present invention provides an antibody conjugate according to the first aspect of the invention for use in the treatment of T-cell lymphoma or leukemia.

[0027] Treatment of T cell lymphoma or leukemia in a subject can 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 the malignant T cells, but not normal T cells that express TRBCs that are not expressed by the malignant T cells.

[0028] The method may further include examining the TCR β chain constant region (TCRB) of malignant T cells from the subject to determine whether the malignant T cells from the subject express TRBC1 or TRBC2.

[0029] The T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0030] In a third aspect, the present invention provides an antibody conjugate according to an embodiment of the first aspect of the invention for use in a method for targeting the delivery of a chemotherapeutic agent to cells expressing TRBCs 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 invention and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0032] In a fifth aspect, the present invention provides a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, comprising: i) determining whether malignant T cells in a sample isolated from a subject express TRBC1 or TRBC2; ii) selecting an antibody conjugate for use according to the second aspect of the invention based on TRBC1 or TRBC2 expression on malignant T cells; The present invention provides a method comprising:

[0033] In a 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 invention, comprising the steps of: i) determining whether malignant T cells in a sample isolated from a subject express TRBC1 or TRBC2; ii) selecting a subject to receive a therapy based on the antibody conjugate for use according to the second aspect of the invention based on TRBC1 or TRBC2 expression on malignant T cells; The present invention provides a method comprising: [Brief explanation of the drawings]

[0034] [Figure 1] Schematic of the αβ T cell receptor / CD3 complex. The T cell receptor is formed from six distinct protein chains that must be assembled in the endoplasmic reticulum to be expressed on the cell surface. Four proteins of the CD3 complex (CD3ζ, CD3γ, CD3ε, and CD3δ) coat the T cell receptor (TCR). The TCR confers specificity to the complex for a particular antigen and is composed of two chains, TCRα and TCRβ. Each TCR chain has a membrane-distal variable component and a membrane-proximal constant component. Nearly all T cell lymphomas and many T cell leukemias express the TCR / CD3 complex. [Figure 2]Separation of T cell receptor beta constant region (TRBC)-1 and TRBC2 during T cell receptor rearrangement. Each TCR beta chain is formed from the genomic recombination of specific beta variable (V), diversity (D), joining (J), and constant (TRBC) regions. The human genome contains two highly similar and functionally equivalent TRBC loci, known as TRBC1 and TRBC2. During TCR gene rearrangement, the J region recombines with either TRBC1 or TRBC2. This rearrangement is permanent. T cells express many copies of a single TCR on their surface; therefore, each T cell expresses a TCR whose beta 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] Internalization 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 performed on various mutated antibody variants to determine affinity, association and dissociation rates [Figure 5-2] Surface plasmon resonance experiments performed on various mutated antibody variants to determine affinity, association and dissociation rates [Figure 5-3] Surface plasmon resonance experiments performed on various mutated antibody variants to determine affinity, association and dissociation rates [Figure 5-4] Surface plasmon resonance experiments performed on various mutated antibody variants to determine affinity, association and dissociation rates [Figure 6] Analysis of the binding kinetics of mutated antibody variants. Several binders were identified that had very similar association rates but different dissociation rates. [Figure 7]Internalization profiles of pHrodo Green-conjugated variants of aTRBC1 in HPB-ALL TRBC1 and KO cells at 9 hours. Clones were selected with increasing dissociation rates for TRBC1. Flow cytometry demonstrates improved internalization of binders with faster dissociation rates, up to Mut13, before internalization is affected by faster off-rates. The antibody variants are listed in Tables 1 and 2. [Figure 8] Internalization 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 an MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the ability of the antibodies to be internalized. MFI: mean fluorescence intensity. C. Cytotoxicity assay of MMAE-conjugated Mut11, Mut15, and anti-HEL against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO. ****P<0.0001 by comparison of fit to 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 an MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the ability of the antibodies to be internalized. MFI: mean fluorescence intensity. C. Cytotoxicity assay of MMAE-conjugated Mut11, Mut15, and anti-HEL against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO. ****P<0.0001 by comparison of fit to 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 an MC-valine-citrulline linker. B. Conjugation of Mut11 and Mut15 antibodies to monomethyl auristatin E (MMAE) did not impair the ability of the antibodies to be internalized. MFI: mean fluorescence intensity. C. Cytotoxicity assay of MMAE-conjugated Mut11, Mut15, and anti-HEL against HPB-ALL TRBC1+, HPB-ALL TRBC2+, and HPB-ALL TCR KO. ****P<0.0001 by comparison of fit to 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). DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention The present invention provides antibody conjugates that specifically bind to the TCR β chain constant region (TRBC), which have excellent internalization properties 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. Binding of the TCR to an antigenic peptide and MHC (peptide / MHC) leads to T lymphocyte activation through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

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

[0038] Each α and β chain is composed of two extracellular domains: a variable (V) region and a constant (C) region, both of which are immunoglobulin superfamily (IgSF) domains that form an antiparallel β-sheet. The constant region is adjacent 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 connective sequence in which cysteine ​​residues form disulfide bonds, forming the link between the two chains.

[0039] The variable domains of both the TCR α and β chains have three hypervariable or complementarity determining regions (CDRs). The variable region of the β chain has an additional hypervariable region (HV4), which does not normally contact 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 through 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 paired assembly of the subunits, in which both the transmembrane and extracellular domains of TCRα and β and CD3γ and δ play a role.

[0041] Thus, a TCR generally consists of a CD3 complex and TCR α and β chains, which in turn consist of variable and constant regions (FIG. 1).

[0042] The locus (Chr7:q34) supplying the TCR β chain constant region (TRBC) has duplicated over evolutionary history 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 therefore each αβ T cell expresses either TRBC1 or TRBC2 in a mutually exclusive manner.

[0043] The present inventors have previously determined that it is possible to distinguish between TRBC1 and TRBC2 despite the similarity between their sequences. The present inventors have also previously determined that it is possible to distinguish between the amino acid sequences of TRBC1 and TRBC2 while they are present in situ on the surface of cells, such as T cells (WO 2015132598). Furthermore, numerous antibodies specific for either TRBC1 or TRBC2 have been produced (WO 2015132598).

[0044] 2. Antibody Conjugates In a first aspect, the present invention provides an antibody conjugate, hereinafter "antibody conjugate of the present invention", that specifically binds to a TCR β chain constant region (TRBC), wherein the antibody binds to a TCR β chain constant region (TRBC) within 0.001 seconds. -1 ~0.5 seconds -1 The dissociation rate constant (k d )

[0045] The term "antibody conjugate" as used herein refers to a compound comprising an antibody linked to a therapeutic entity or payload via a chemical linker. Upon binding 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 the cleavable linker (e.g., by cathepsin B found in lysosomes) or by proteolysis of the antibody if linked to the payload via a non-cleavable linker.

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

[0047] The term "antibody" as used herein refers to a polypeptide having an antigen-binding site comprising at least one complementarity-determining region or CDR. An antibody may comprise 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 comprise 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-binding site to bind the antigen.

[0048] A full-length antibody or immunoglobulin typically consists 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, and 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. The variable regions of each pair of light and heavy chains are characterized by the same general structure, composed of relatively conserved regions called frameworks (FRs) connected by three hypervariable regions called complementarity-determining regions (CDRs) (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 th(Ed., NIH Publication No. 91-3242, Bethesda, MD.; Chothia & Lesk, 1987, J Mol Biol 196:901-17). As used herein, the term "complementarity-determining region" or "CDR" refers to the region in an antibody that complements the shape of an antigen. Thus, CDRs determine the affinity and specificity of the protein for a particular antigen. The CDRs of the two chains of each pair are aligned by the framework regions and acquire the function of binding 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 CDRs are in common use. The Kabat definition is based on sequence variability and 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 determining regions (CDR-IMGT) are the loop regions of the variable domain, delimited according to the IMGT specific numbering for the V domain. There are three CDR-IMGTs in the variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C''), and CDR3-IMGT (loop FG). Other definitions of CDRs have also been developed, such as the Chothia, AbM, and contact definitions (see http: / / www.imgt.org).

[0050] The antibody conjugates of the present invention may comprise a full-length antibody or an antigen-binding fragment thereof.

[0051] The antibody conjugate of the present invention may comprise 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 portion" 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. An antibody fragment may include, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, a Fab fragment, a F(ab')2 fragment, an Fv fragment, a single-chain Fv (scFv), a domain antibody (dAb or VH), a single-domain antibody (sdAb), a VHH, a nanobody, a diabody, a triabody, a trimeric body, and a monobody.

[0053] The antibody conjugate of the present invention can comprise an antigen-binding domain based on a non-immunoglobulin scaffold. These antibody-binding domains are also called antibody mimics. Non-limiting examples of non-immunoglobulin antigen-binding domains include affibodies, fibronectin artificial antibody scaffolds, anticalins, affilins, DARPins, VNARs, iBody, affimers, finomers, abdulins / nanoantibodies, centilins, alphabodies, nanophytins, and D domains.

[0054] The antibody may be bifunctional.

[0055] Antibodies may be non-human, e.g., murine, rat, or camelid, chimeric, humanized, or fully human. Antibodies may be synthetic.

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

[0057] The antibody used in the antibody conjugate of the present invention specifically binds TRBC. The ability of the antigen-binding domain to specifically bind TRBC 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 can bind either TRBC1 or TRBC2. Methods for determining whether an antibody is specific for TRBC1 or TRBC2 are described in WO 2015 / 132598.

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

[0060] The terms "internalization" and "cellular uptake" are used interchangeably in the context of the present invention, and as used herein, refer to a process also known as receptor-mediated endocytosis.Endocytosis is a cellular process in which molecules or substances are transported into cells through cell membrane encapsulation.After internalization, antibody-conjugated molecules are transported to lysosomes, where their cargo is released.Therefore, the speed and extent of antibody-conjugate internalization are crucial to the efficacy of the antibody-conjugate.

[0061] A number of techniques, including microscopy and flow cytometry, can be used to identify antibodies with the desired cellular uptake rate. Immunofluorescence microscopy-based colocalization with endosomal proteins can also be used to monitor cellular uptake. Unlike immunofluorescence microscopy, flow cytometry using fluorescently labeled antibodies allows for a more rapid and quantitative assessment of antibody internalization, potentially allowing for higher throughput. In flow cytometry-based assays, a fluorescently labeled secondary antibody can be used to measure how much antibody remains surface-bound after an incubation period. Alternatively, cells can be treated with a fluorescently labeled primary antibody prior to quenching cell surface fluorescence with an anti-fluorophore antibody. Given that ADCs require endocytosis and subsequent acidification to be effective, pH-sensitive labels are extremely useful for tracking the internalization 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 dye, which exhibits very low fluorescence at neutral pH and increases fluorescence as the pH becomes more acidic. Methods for labeling antibodies with fluorescent dyes are well known in the art.

[0062] The term "affinity" as used herein refers to the strength of the interaction between the antigen-binding site of an antibody and an epitope. Affinity is usually measured by the dissociation rate constant (k d or k off ) and the association rate constant (k aor k on ), i.e., k d / k a or k off / k on The equilibrium dissociation constant (KD) is measured as the equilibrium dissociation constant (KD), where KD is the on-rate constant, and KD and affinity are inversely related. As used herein, "association rate constant" or "on-rate" or "k a " or "k on The term "dissociation rate constant" or "off-rate" or "k" refers to a constant used to characterize how rapidly an antibody binds to its target. d " or "k off The term "relative binding" 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 is measured in k d is seconds -1 It is measured in

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

[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 carried out using various commercially available instruments using different parameters.For example, the KD and kinetic rate constant can be measured on a Biacore T200 instrument at a flow rate of 30 ml / min at 25 °C using HBSP1 as running and dilution buffer (GE Healthcare BioSciences).The kinetic rate constant can be obtained by curve fitting according to the 1:1 Langmuir binding model.

[0065] The inventors have found that antibodies-1 ~0.3 seconds -1 Dissociation rate constant k in the range d It has been determined that antibodies for use in the antibody conjugates of the present invention exhibit optimal internalization properties when they have a solubility of 0.001 seconds. -1 ~0.25 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.001 seconds. -1 ~0.2 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.001 seconds. -1 ~0.15 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.001 seconds. -1 ~0.1 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.002 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a denaturation rate of 0.003 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.004 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.005 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.006 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.007 seconds. -1 ~0.3 seconds -1 k in the range dThe antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.008 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a viscosity of 0.009 seconds. -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a molecular weight of 0.01 s -1 ~0.3 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a molecular weight of 0.01 s -1 ~0.2 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a molecular weight of 0.01 s -1 ~0.15 seconds -1 k in the range d The antibodies for use in the antibody conjugates of the invention may have a molecular weight of 0.01 s -1 ~0.1 seconds -1 k in the range d The antibody may have a viscosity of 0.002 seconds. -1 ~0.1 seconds -1 k in the range d 2. The antibody conjugate of claim 1, wherein the antibody has a solubility of 0.017 seconds. -1 ~0.083 seconds -1 k in the range d 2. The antibody conjugate of claim 1, having:

[0066] Antibodies for use in the antibody conjugates of the present invention may be used in concentrations of 1 x 10 2 M -1 ~1×10 6 M -1 The association rate constant (k a The antibody for use in the antibody conjugate of the invention may further comprise 5 x 10 2 M -1 ~5×10 5 M -1 k in the range aThe antibody for use in the antibody conjugate of the invention may further have a concentration of 1 x 10 3 M -1 ~5×10 5 M -1 k in the range a The antibody for use in the antibody conjugate of the invention may further have a concentration of 5×10 3 M -1 ~5×10 5 M -1 k in the range a The antibody for use in the antibody conjugate of the invention may further have a concentration of 1 x 10 4 M -1 ~1×10 5 M -1 k in the range a The antibody for use in the antibody conjugate of the invention may further have a concentration of 5×10 4 M -1 ~1×10 5 M -1 k in the range a may further comprise:

[0067] Antibodies for use in the antibody conjugates of the present invention may be used in concentrations of 1 x 10 ー10 M~1×10 ー5 Antibodies for use in the antibody conjugates of the invention may further have an affinity constant (KD) in the range of 1 x 10 -10 M~5×10 -6 Antibodies for use in the antibody conjugates of the invention may further have a KD in the range of 5 x 10 -10 M~1×10 -6 Antibodies for use in the antibody conjugates of the invention may further have a KD in the range of 5 x 10 -10 M~5×10 -7 Antibodies for use in the antibody conjugates of the invention may further have a KD in the range of 1 x 10 -9 M~5×10 -7 Antibodies for use in the antibody conjugates of the invention may further have a KD in the range of 5 x 10 -9 M~1×10 -7It may further have a KD in the range of M.

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

[0069] An antibody for use in an antibody conjugate of the invention may have increased internalization upon binding to a target cell compared to the internalization of a reference antibody having a VH domain having the sequence set forth in SEQ ID NO:1 and a VL domain having the sequence set forth in SEQ ID NO:2.

[0070] The internalization of an antibody conjugate of the invention upon binding to a target cell may be increased over that of a reference antibody. The internalization of the antibody conjugate upon binding to a target cell may be increased over that of the reference antibody 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%. Methods for determining and quantifying internalization 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 for TRBC1, has the following mutations or combinations of mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: -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 may include one of:

[0073] The antibody for use in the antibody conjugate of the present invention that is specific for TRBC1 comprises a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2, 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:

[0074] The antibody for use in the antibody conjugate of the present invention that is specific for TRBC1 comprises the following mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: -G106A in the VH domain may include:

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

[0076] These specific combined mutations were shown to alter binding to TRBC1 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 can specifically bind to TRBC2.

[0078] The antibody for use in the antibody conjugate of the present invention that is specific for TRBC2 comprises the following combination of mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: - 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 may include one of:

[0079] An antibody for use in the antibody conjugate of the present invention that is specific for TRBC2 comprises a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2, 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:

[0080] These specific combined mutations were shown to alter binding to TRBC2 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 that is specific for TRBC2 comprises the following mutations compared to a reference antibody having a VH domain having the sequence shown in SEQ ID NO: 1 and a VL domain having the sequence shown in SEQ ID NO: 2: -T28K, Y32F, A100N and N103L in the VH domain and N35K in the VL domain may include:

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

[0083] As used herein, the term "reference antibody" refers to the humanized JOVI-1 antibody, i.e., hJOVI-1, comprising a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2. Murine JOVI-1 has been previously described by Viney et al. (Viney et al., 1992, Hybridoma, 11:701-13). [ka] [ka]

[0084] An antibody for use in an antibody conjugate of the invention may comprise 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.

[0085] Mutants or variants of antibodies for use in the antibody conjugates of the present invention that maintain their specificity and internalization properties also form part of the present invention. As used herein, the term "mutant" or "variant" refers to a polypeptide that differs from a specifically listed polypeptide, i.e., a reference or parent polypeptide, by amino acid insertion, deletion and / or substitution, for example, produced using recombinant DNA technology or by de novo synthesis. Mutants and variants are used interchangeably in the context of the present invention.

[0086] 2.2. Therapeutic Entity or Payload The antibody conjugates of the present invention comprise an antibody bound to a therapeutic entity or payload, which can be displaced by a detection entity for the purpose of investigating the internalization properties of the antibody conjugate.

[0087] The term "therapeutic entity" or "payload" as used herein refers to any molecule that inhibits or suppresses the function of a cell and / or causes destruction of the cell (cell death) and / or exerts an anti-proliferative effect. The payload can be a drug or a radionuclide.

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

[0089] The term "chemotherapeutic entity" as used herein refers to any molecule that inhibits or suppresses cellular function, and / or causes cell death, and / or exerts an anti-proliferative effect. Hereinafter, the resulting conjugate is referred to as an "antibody-drug conjugate (ADC) of the present invention." The chemotherapeutic entity can be a cytotoxic drug or cytotoxin. Numerous classes of cytotoxic agents, including, but not limited to, tubulin inhibitors, DNA damaging 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: the vinca alkaloid-binding site, the taxane-binding site, the colchicine-binding site, the maytansine-binding site, and the laulimalide-binding site. Microtubule / tubulin inhibitors can be classified into two major categories according to their mechanism of action: agents that promote tubulin polymerization and stabilize the microtubule structure (agents that bind to the taxane-binding site and the laulimalide-binding site) and agents that inhibit tubulin polymerization and destabilize the microtubule structure (agents that bind to the vinca alkaloid-binding site, the maytansine-binding site, and the colchicine-binding site). Non-limiting examples of tubulin inhibitors that bind to the vinca alkaloid binding site include vincristine, vinblastine, vinflunine, halichondrin B, eribulin mesylate, cryptophycin, and dolastatins, such as auristatin MMAF, MMAE, PF-06390101, MMAD, auristatin E, auristatin W analogs, auristatin f-HPA, amberstatin 269, and AGD-0182. Non-limiting examples of tubulin inhibitors that bind to the maytansine binding site include maytansine and maytansinoids, such as DM1 and DM4. Non-limiting examples of tubulin inhibitors that bind to the colchicine binding site include colchicine, 2-methoxyestradiol, sulfonamides, and Aspergillus derivatives. Non-limiting examples of tubulin inhibitors that bind to the taxane binding site include paclitaxel, docetaxel, cyclostreptin, eluterobin, ABI-007, ixabepilone, patupilone, and BMS-310705. Non-limiting examples of tubulin inhibitors that bind to the laulimalide binding site include laulimalide and peloruside A.

[0091] DNA damaging agents include, but are not limited to, calicheamicins (ozogamicins), such as calicheamicin γ1; pyrrolobenzodiazepines, such as PDB taliline and SG3249; duocarmycins, such as DUBA; camptothecin analogs, such as SN38 and DX-8951; anthracyclines; and doxorubicin (adriamycin). Also included are alkylating agents, nitrosoureas, ethyleneimine / methylmelamine, alkylsulfonates, antimetabolites, pyrimidine analogs, epipodophyllotoxins, platinum coordination 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] Suitable cytotoxins for use in ADCs are also described, for example, in WO 2015 / 155345 and WO 2015 / 157592.

[0095] Chemotherapeutic entities include biological response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; substituted ureas such as anthracenediones, hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including corticosteroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide; progestins such as hydroxyprogesterone acetate, medroxyprogesterone acetate, and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolide; and nonsteroidal antiandrogens such as flutamide.

[0096] The antibody in the antibody conjugate of the present invention may be conjugated to a radionuclide. Hereinafter, the resulting conjugate is referred to as the "antibody-radionuclide conjugate (ARC) of the present invention." Radioimmunoconjugates have unique theranostic (i.e., therapeutic and diagnostic) potential. For diagnostic purposes, the antibody may be labeled with a radionuclide compatible with imaging procedures such as single-photon emission computed tomography or positron emission tomography (PET). For therapeutic purposes, the choice of radionuclide largely depends 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 contemplated by the present invention. One of the main attractive features of radioimmunotherapy is the crossfire or bystander effect, i.e., the ability to damage cells in close proximity to the site of antibody localization. In most cases, radiolabeling of antibodies is achieved by tyrosine iodination or by conjugation of metal chelators, such as diethylenetriaminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), to the antibody molecule.

[0097] The detectable entity can be a fluorescent entity, such as a fluorescent peptide, dye, or label. As used herein, the term "fluorescent entity" refers to a moiety 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 internalization 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 dye, which exhibits very low fluorescence at neutral pH and increases fluorescence as the pH becomes more acidic.

[0098] The antibody conjugates of the invention conjugated to a detectable entity can be used to determine the TRBC of malignant T cells.

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

[0100] 2.3. Conjugation Chemistry The antibody conjugates of the present invention comprise an antibody linked via a conjugation or chemical linker to a chemotherapeutic entity, a radionuclide, or a detection entity.

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

[0102] When 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.The position on the Fc region suitable for site-specific conjugation is well known in the art.Alternatively, the payload can be conjugated to the antibody via thiol-maleimide bond in the hinge and heavy and light chains.

[0103] Chemical linkers must be sufficiently stable in the systemic circulation and capable of rapidly and efficiently releasing the cytotoxic drug upon internalization of the antibody conjugate within cancer cells. According to the drug release mechanism, linkers for antibody conjugates are generally classified into cleavable linkers (acid-labile 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 the cytotoxic drug, whereas antibody conjugates with cleavable linkers require hydrolysis, enzymatic reaction, or reduction to selectively release the cytotoxic drug based on the physiological environment to which the antibody conjugate is exposed. [Table 3]

[0104] Three main types of cleavable linkers are often used in ADCs: acid-cleavable linkers, protease-cleavable linkers, and disulfide linkers. Acid-cleavable linkers, such as hydrazine linkers (AcBut), are designed to be stable at the neutral pH of the circulation but can be hydrolyzed in the low-pH environment of lysosomes. Protease-cleavable linkers are also used to keep antibody conjugates intact in the systemic circulation and allow for the easy release of cytotoxic drugs from the antibody conjugate by lysosomal enzymes in cancer cells. For example, valine-citrulline (Val-Cit) and phenylalanine-lysine (Phe-Lys) confer 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 drug into cells. Antibody conjugates with reducible disulfide linkers generate uncharged metabolites that can diffuse to neighboring cells and induce bystander killing, which is advantageous for killing heterogeneous tumors.

[0105] A non-limiting example of a non-cleavable 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 Compositions In another aspect, the present invention also relates to a pharmaceutical composition comprising the antibody conjugate of the present invention, hereinafter "the pharmaceutical composition of the present invention".

[0108] Pharmaceutical composition can further comprise pharmaceutically acceptable carrier, diluent, excipient or adjuvant.Pharmaceutical composition can optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds.Such preparation can be, for example, suitable for intravenous injection.

[0109] The term "antibody conjugate of the invention" has been described in detail in connection with the above aspect of the invention, and its features and embodiments apply equally to this aspect of the invention.

[0110] Administration Administration of the antibody conjugates of the invention can be achieved using any of a variety of routes that make the active ingredient bioavailable. For example, the agents can be administered via oral and parenteral routes, intraperitoneal, intravenous, subcutaneous, transdermal, intramuscular, or local delivery, e.g., by catheter or stent.

[0111] Typically, a physician will determine the actual dosage most suitable for an individual subject, and the actual dosage will vary with the age, weight, and response of the particular patient. The dosage will be 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 of the present invention for use in medicine.

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

[0114] This aspect of the invention may alternatively be formulated as an antibody conjugate of the invention for use in the treatment of T-cell lymphoma or leukemia, hereinafter "antibody conjugate for use of the invention".

[0115] This aspect of the invention may alternatively be formulated as the use of an antibody conjugate of the invention in the manufacture of a medicament for treating T-cell lymphoma or leukemia.

[0116] The term "antibody conjugate of the invention" has been described in detail in connection with the above aspects of the invention, and its features and embodiments apply equally to these aspects of the invention.

[0117] Methods for treating T-cell lymphoma and / or leukemia relate to therapeutic uses of the antibody conjugates of the invention that may be administered to a subject with an existing T-cell lymphoma and / or leukemia to alleviate, reduce or ameliorate at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0118] A method for preventing T-cell lymphoma and / or leukemia relates to the prophylactic use of the antibody conjugate of the present invention. Herein, such an antibody conjugate can be administered to a subject who has not yet suffered from T-cell lymphoma and / or leukemia and / or who does not show any symptoms of T-cell lymphoma and / or leukemia to suppress or reduce the cause of the disease or to alleviate or prevent the occurrence of at least one symptom associated with the disease. The subject may have a predisposition to T-cell lymphoma and / or leukemia or may be considered at risk for developing T-cell lymphoma and / or leukemia.

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

[0120] Treatment of T cell lymphoma or leukemia in a subject can 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 the malignant T cells, but not normal T cells that express TRBCs that are not expressed by the malignant T cells.

[0121] The term "subject" or "individual" as used in the context of the present invention refers to a member of a mammalian species, preferably a human being, male or female, of any age or race.

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

[0123] As used herein, the term "therapeutically effective amount" refers to the amount of an antibody conjugate of the invention required to achieve appreciable prevention, cure, delay, reduction in severity, or amelioration of one or more symptoms of T-cell lymphoma and / or leukemia.

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

[0125] The methods of the invention can be used to treat T-cell leukemia. "Leukemia" is used herein according to its standard meaning to refer to a cancer of the blood or bone marrow.

[0126] The following is an exemplary, non-exhaustive 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's lymphomas (NHLs). However, peripheral T-cell lymphomas are associated with an aggressive clinical course, and the cause and precise cellular origin of most T-cell lymphomas remain ill-defined.

[0128] Lymphoma usually first appears as swelling in the neck, armpits, or groin. Additional swelling may occur if other lymph nodes, such as the spleen, are located nearby. Enlarged lymph nodes typically invade blood vessels, nerves, or the stomach, resulting in swelling of the arms and legs, tingling and numbness, or a feeling of fullness, respectively. Lymphoma symptoms also include nonspecific symptoms such as fever, chills, unexplained weight loss, night sweats, lethargy, and itching.

[0129] To delineate prognostically and therapeutically meaningful classifications for peripheral T-cell lymphomas, the WHO classification utilizes morphological and immunophenotypic features in conjunction with clinical and, in some cases, genetic information (Swerdlow et al., WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, 4th ed.; Lyon: IARC Press; 2008). The anatomical location of neoplastic T cells partially resembles their proposed normal cellular counterparts and functions, and thus T-cell lymphomas are associated with lymph nodes and peripheral blood. This approach allows for a better understanding of some of the manifestations of T-cell lymphomas, including their cellular distribution, some aspects of morphology, and associated clinical findings.

[0130] The most common type of T-cell lymphoma is peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), accounting for 25% of the total, followed by angioimmunoblastic T-cell lymphoma (AITL) (18.5%).

[0131] Peripheral T-cell lymphoma, nonspecific (PTCL-NOS) PTCL-NOS accounts for more than 25% of all peripheral T-cell lymphomas and NK / T-cell lymphomas and is the most common subtype. PTCL-NOS is a diagnosis of exclusion and does not correspond to any of the specific mature T-cell lymphoma entities listed in the current WHO 2008 guidelines. Therefore, PTCL-NOS resembles diffuse large B-cell lymphoma, not otherwise specified (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 are of lymph node origin, but approximately 13% of patients develop extranodal disease, most commonly involving 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 lymphoepithelioid (Lennert) variant, T-zone variant, and follicular variant. The lymphoepithelioid variant of PTCL contains abundant background epithelioid 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 have 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 almost always downregulated. CD30 and, rarely, CD15 may be expressed, with CD15 being an adverse prognostic feature. CD56 expression, although rare, also has a negative prognostic impact. Additional adverse pathological prognostic factors include a proliferation rate of greater than 25% based on KI-67 expression and the presence of greater than 70% transformed cells. Immunophenotypic analysis of these lymphomas has provided little insight into their biology.

[0135] Angioimmunoblastic T-cell lymphoma (AITL) AITL is a systemic disease characterized by perivascular expansion of lymph nodes with polymorphic infiltrates, prominent high endothelial venules (HEVs), and follicular dendritic cell (FDC) meshwork. AITL is considered a de novo T-cell lymphoma derived from alpha-beta T cells of the follicular helper type (TFH), usually found in germinal centers.

[0136] AITL is the second most common entity among peripheral T-cell lymphoma and NK / T-cell lymphoma, accounting for approximately 18.5% of cases. AITL occurs in middle-aged to older adults, with a median age of 65 years, and is approximately equally prevalent in men and women. Clinically, patients usually have advanced-stage disease with generalized lymphadenopathy, hepatosplenomegaly, and prominent systemic symptoms. A pruritic skin rash is commonly present. Polyclonal hypergammaglobulinemia associated with autoimmune phenomena is often present.

[0137] Three distinct morphologic patterns of AITL have been described. The early lesions of AITL (pattern I) usually show a preserved architecture with characteristic hyperplastic follicles. The neoplastic proliferation is localized around the follicles. In pattern II, the nodular architecture is partially lost, with little retention of atretic follicles. The marginal sinuses are preserved and even dilated. The paracortex contains branching HEVs, and there is proliferation of FDCs overlying the B-cell follicles. The neoplastic cells are small to medium in size, with minimal cytologic atypia. The neoplastic cells often have clear to pale cytoplasm and may show distinct T-cell membranes. A polymorphic inflammatory background is usually evident.

[0138] Although AITL is a T-cell malignancy, there is a characteristic expansion of B cells and plasma cells, which may reflect the function of the neoplastic cells as TNF-α cells. Both EBV-positive and EBV-negative B cells are present. Occasionally, atypical B cells can morphologically and immunophenotypically resemble Hodgkin / Reed-Sternberg-like cells, occasionally leading to diagnostic confusion with that entity. B-cell proliferation in AITL can be extensive, and some patients develop secondary EBV-positive diffuse large B-cell lymphoma (DLBCL) or, more rarely, EBV-negative B-cell neoplasms, often with plasmacytic differentiation.

[0139] Neoplastic CD4+ T cells in AITL show strong expression of CD10 and CD279 (PD-1) and are positive for CXCL13. CXCL13 leads to increased B cell recruitment to lymph nodes via HEV attachment, B cell activation, plasma cell differentiation, and expansion of FDC meshworks, all of which contribute to the morphological and clinical characteristics 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 separate entity with a TFH phenotype. In contrast to AITL, the follicular variant of PTCL-NOS does not have prominent HEV or extrafollicular extension of FDC networks. The neoplastic cells form intrafollicular aggregates and may mimic B-cell follicular lymphoma, but they may also have an interfollicular growth pattern or an expanded mantle zone. Clinically, the follicular variant of PTCL-NOS differs from AITL because patients more often present with early-stage disease with partial lymph node involvement and may lack the systemic symptoms associated with AITL.

[0141] Anaplastic large cell lymphoma (ALCL) ALCL can be subdivided as 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 metastases to extranodal sites (skin, bone, soft tissue, lung, liver) and B syndromes are common.

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

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

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

[0146] ALCL-ALK- was included as a provisional category in the 2008 WHO classification. ALCL-ALK- is defined as a CD30-positive T-cell lymphoma with an adhesive growth pattern and the presence of hallmark cells but lacking ALK protein expression, making it morphologically indistinguishable from ALCL-ALK+.

[0147] In contrast to ALCL-ALK+, which is more common in children and young adults, patients are usually adults between the ages of 40 and 65. ALCL-ALK- can be present in both lymph nodes and extralymphatic tissue, although the latter is less common in ALCL-ALK+. Most cases of ALCL-ALK- show obliteration of lymph node architecture by sheets of cohesive neoplastic cells with typical "hallmark" features. In contrast to ALCL-ALK+, the small cell morphology variant is not observed.

[0148] Unlike its ALK+ counterpart, ALCL-ALK- shows greater conservation of surface T-cell marker expression, but less frequent expression of cytotoxicity markers and epithelial membrane antigen (EMA). Gene expression signatures and recurrent chromosomal imbalances differ in 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 marked differences in prognosis between these three distinct entities. The 5-year overall survival rate for ALCL-ALK- has been reported to be 49%, which is not as good as the 5-year overall survival rate for ALCL-ALK+ (70%), but is also significantly better than the 5-year overall survival rate for PTCL-NOS (32%).

[0150] Enteropathy-associated T-cell lymphoma (EATL) EATL is an aggressive neoplasm thought to originate from intestinal intraepithelial T cells. Two morphologically, immunohistochemically, and genetically distinct types of EATL are recognized in the 2008 WHO classification: type I (representing the majority of EATL) and type II (accounting for 10–20% of cases).

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

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

[0153] The cytological spectrum of EATL type I is broad, and some cases may contain undifferentiated cells. A polymorphic inflammatory background is present, which may obscure the neoplastic component in some cases. The intestinal mucosa in areas adjacent to the tumor often exhibits features of celiac disease, with blunting of the villi and an increased number of intraepithelial lymphocytes (IELs), which may represent lesion precursor cells.

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

[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 within the mucosa is often present, often without an inflammatory background. Most cases express the gamma delta TCR, although cases associated with the alpha beta TCR do exist.

[0156] Type II EATL is more widespread worldwide than type I EATL and is often seen in Asian or Latin American populations, where celiac disease is rare. In individuals with European ancestry, type II represents approximately 20% of intestinal T-cell lymphomas, and at least a subset of cases have a history of celiac disease. The clinical course is aggressive.

[0157] Hepatosplenic T-cell lymphoma (HSTL) HSTL is an aggressive, systemic neoplasm generally derived from gamma-delta cytotoxic T cells of the innate immune system, although in rare cases it can also originate from alpha-beta T cells. HSTL is one of the rarest T-cell lymphomas and typically affects adolescents and young adults (median age 35 years) with a strong male predominance.

[0158] Extranodal NK / T cell lymphoma nasal type Extranodal NK / T-cell lymphoma, nasal type, is an aggressive disease often associated with destructive midline lesions and necrosis. Most cases are of NK cell origin, but some cases are derived from cytotoxic T cells. Extranodal NK / T-cell lymphoma, nasal type, is commonly associated with Epstein-Barr virus (EBV).

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

[0160] Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells to the skin, resulting in the appearance of various lesions that change shape as the disease progresses, typically beginning as what looks like a rash 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-exhaustive list: mycosis fungoides, Pagetoid reticulosis, Sézary syndrome, granulomatous lax cutis, lymphomatoid papulosis, pityriasis lichenoides chronica, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30- cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, and angiocentric lymphoma.

[0162] Signs and symptoms of CTCL vary depending on the specific disease, the two most common types of which are mycosis fungoides and Sézary syndrome. Classical mycosis fungoides is divided into three stages. -Erythema (atrophic or nonatrophic): nonspecific dermatitis, erythema of the lower trunk and buttocks; minimal / absent pruritus; - Plaques: intensely pruritic plaques, lymphadenopathy; and Tumor: Ulcerative tendency

[0163] Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymphadenopathy, palmar and / or plantar hyperkeratosis, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly.

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

[0165] The primary pathogenic mechanism for the development of cutaneous T-cell lymphoma (i.e., mycosis fungoides) is unknown. Mycosis fungoides can be preceded by a T-cell-mediated chronic inflammatory skin disease that can occasionally progress to a fatal lymphoma.

[0166] Primary cutaneous ALCL (C-ALCL) C-ALCL is often indistinguishable from ALC-ALK- by morphology. C-ALCL is defined as a large-cell skin tumor with undifferentiated, pleomorphic, or immunoblastic morphology in which more than 75% of cells express CD30. C-ALCL belongs to the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders, which, together with lymphomatoid papulosis (LyP), constitute the second most common group of cutaneous T-cell lymphoproliferative disorders following mycosis fungoides.

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

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

[0169] The methods of the present invention can be used to treat T-ALL associated with malignant T cells expressing TCRs, including TRBCs.

[0170] T-cell prolymphocytic leukemia T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell leukemia with aggressive behavior and a tendency to metastasize to the blood, bone marrow, lymph nodes, liver, spleen, and skin. T-PLL primarily affects adults over the age of 30. Other names include T-cell chronic lymphocytic leukemia, the "knob-like" form of T-cell leukemia, and T-prolymphocytic / T-cell lymphocytic leukemia.

[0171] In peripheral blood, T-PLL consists of medium-sized lymphocytes with a single nucleus and basophilic cytoplasm with occasional blebs or projections. The nuclei are usually round to oval in shape, but occasionally patients have cells with a more irregular nuclear contour, resembling the convoluted nuclear shape seen in Sézary syndrome. The small cell variant constitutes 20% of all T-PLL cases, while the Sézary cell-like (convoluted) variant is seen in 5% of cases.

[0172] T-PLL has the immunophenotype of mature (postthymic) T lymphocytes, and the neoplastic cells are typically positive for the pan-T antigens CD2, CD3, and CD7, and negative for TdT and CD1a. The immunophenotype CD4+ / CD8- is present in 60% of cases, the CD4+ / CD8+ immunophenotype is present in 25%, and the CD4- / CD8+ immunophenotype is present in 15% of cases.

[0173] The T-cell lymphoma or leukemia to be treated or prevented may be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0174] The method of treatment may comprise administering an antibody conjugate of the present invention. One skilled in the art would be able to determine by conventional methods the amount of the antibody conjugate of the present invention that is capable of exerting a therapeutic effect on a patient.

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

[0176] The term "antibody conjugate of the invention" has been described in detail in connection with the above aspect of the invention, and its features and embodiments apply equally to this aspect of the invention.

[0177] 5. Personalized medicine methods Because T cell malignancies are clonal, all malignant cells express either TRBC1 or TRBC2. The present inventors have previously demonstrated that immunotherapy targeting either TRBC1 or TRBC2 offers the ability to treat T cell lymphoma while potentially providing an acceptable toxicity profile (Maciocia et al., 2017, Nat Med 23:1416-23; WO 2015 / 132598). The present invention also provides a method for identifying a subject with 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 sample containing T cells from the subject.

[0178] Thus, in another aspect, the present invention provides a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, comprising: i) determining whether malignant T cells in a 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, hereinafter referred to as "first method of personalized medicine of the present invention," comprising:

[0179] The terms "antibody conjugate of the invention" and "subject" have been described in detail in connection with the above aspect of the invention, and their features and embodiments apply equally to this aspect of the invention.

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

[0181] In another 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 present invention, comprising the steps of: i) determining whether malignant T cells in a sample isolated from the subject express TRBC1 or TRBC2; ii) selecting the subject for therapy based on the 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, hereinafter referred to as the "second personalized medicine method of the present invention," comprising:

[0182] The terms "antibody conjugate of the invention" and "subject" have been described in detail in connection with the above aspect of the invention, and their features and embodiments apply equally to this aspect of the invention.

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

[0184] In the first and second personalized medicine methods of the present invention, a sample containing T cells, such as a biological sample, is obtained from the subject to be tested. As used herein, the term "sample" or "biological sample" includes different types of biological fluids or tissue sections from the affected organ containing T cells. 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, lymphatic fluid, and spinal fluid. These biological fluid samples can be obtained by any conventional method known to those skilled in the art. Alternatively, the sample can also be a section of tissue sample from the affected organ, such as lymph nodes, spleen, tonsils, or thymus, which can be obtained by any conventional method, such as biopsy or surgical resection, or from frozen sections taken for histological purposes.

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

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

[0187] Several immunological methods are available to determine whether malignant T cells express conventional TRBC1 or TRBC2. Non-limiting examples include immunohistochemistry and flow cytometry.

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

[0189] 6. Other Aspects of the Invention 6.1.TRBC1-specific antibody In a further aspect, the present invention provides an antibody comprising the following combination of mutations in the VH domain compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth 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 present invention provides an antibody that specifically binds to TRBC1, including one of the above, hereinafter referred to as the "TRBC1-specific antibody of the present invention."

[0190] Antibodies for use in the antibody conjugates of the present invention that are specific for TRBC2 may contain 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 together with 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.

[0191] The term "antibody" has been described in detail in relation to the first aspect of the invention, and its features and embodiments apply equally to this aspect of the invention.

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

[0193] 6.2.TRBC2-specific antibodies In a further aspect, the present invention provides an antibody comprising the following mutations in the VH domain compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2: -T28R, Y32F and A100N in the VH domain The present invention provides an antibody that specifically binds to TRBC1, including one of the following, hereinafter referred to as the "TRBC2-specific antibody of the present invention."

[0194] Antibodies for use in the antibody conjugates of the present invention that are specific for TRBC2 may contain the following mutations: -T28R, Y32F and A100N in the VH domain together with 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.

[0195] The term "antibody" has been described in detail in relation to the first aspect of the invention, and its features and embodiments apply equally to this aspect of the invention.

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

[0197] 6.2. Chimeric Antigen Receptors In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a TRBC1-specific antibody of the present invention or a 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" have been described in detail in the previous aspects, and their features and embodiments apply equally to this aspect of the present invention.

[0199] As used herein, the term "chimeric antigen receptor" or "CAR" or "chimeric T cell receptor" or "artificial T cell receptor" or "chimeric immunoreceptor" refers to a chimeric type I transmembrane protein that connects an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). Binders are typically single-chain variable fragments (scFv) derived from monoclonal antibodies (mAbs) but can be based on other formats that contain antigen-binding sites. A spacer domain is usually required to separate the binder from the membrane and allow for proper orientation of the binder. A common spacer domain used is the Fc of IgG1. Depending on the antigen, a more compact spacer, such as a stalk from CD8α or even simply an IgG1 hinge, may be sufficient. The transmembrane domain anchors the protein to the cell membrane and connects the spacer to the endodomain.

[0200] Early CAR designs contained endodomains derived from either the γ chain of FcεR1 or the intracellular portion of CD3ζ. As a result, these first-generation receptors transduced immunological signal 1, which was sufficient to trigger T cell killing of cognate target cells, but failed to fully activate T cells to proliferate and survive. To overcome this limitation, compound endodomains have been constructed; that is, 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 transduce activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, 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 transduce survival signals, have also been described. Even more potent third-generation CARs, with endodomains capable of transducing activation, proliferation, and survival signals, have now been described.

[0201] When a CAR binds a target antigen, it transmits an activation signal to the T cell on which the target antigen is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell to tumor cells expressing the targeted antigen.

[0202] Thus, a CAR typically comprises: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that comprises or is associated with a signaling domain (see Figure 4). CAR has the general structure: It may have an antigen-binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain).

[0203] The CARs of the invention can include a signal peptide so that when the CAR is expressed in a cell, such as a T cell, the nascent protein is targeted to the endoplasmic reticulum and then to the cell surface, where it is expressed.

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

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

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

[0207] The signal peptide of SEQ ID NO: 3 is compact and highly efficient, predicted to give approximately 95% cleavage after the terminal glycine, allowing efficient removal by signal peptidases. 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 contain a spacer sequence that links 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 orient in different directions to facilitate binding.

[0211] In the CAR of the present invention, the spacer sequence can include, for example, an IgG1 Fc region, an IgG1 hinge, or a human or mouse CD8 stalk. Alternatively, the spacer can include an alternative linker sequence with similar length and / or domain spacing characteristics to the IgG1 Fc region, IgG1 hinge, or CD8 stalk. The human IgG1 spacer can be modified to remove the Fc binding motif. The spacer can include a coiled-coil domain, for example, as described in WO 2016 / 151315.

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

[0213] The COMP coiled-coil domain can be truncated at the N-terminus and retain surface expression. Thus, the coiled-coil COMP spacer can comprise or consist of a truncated version of SEQ ID NO: 18 truncated at the N-terminus. Truncated COMP can contain the five C-terminal amino acids of SEQ ID NO: 19, i.e., the sequence CDACG (SEQ ID NO: 20). Truncated COMP can contain 5 to 44 amino acids, e.g., at least 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. Truncated COMP can correspond to the C-terminus of SEQ ID NO: 19. For example, a truncated COMP containing 20 amino acids can contain the sequence QQVREITFLKNTVMECDACG (SEQ ID NO: 21). Truncated COMP can retain the cysteine ​​residues involved in multimerization. Truncated COMP can retain the ability to form multimers.

[0214] The transmembrane domain is the sequence of the CAR that spans the membrane.

[0215] The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the present invention. The presence and total length of a transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, given that the transmembrane domain of a protein has a relatively simple structure, i.e., a polypeptide sequence that is predicted to form a hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM domain can also be used (U.S. Patent No. 7,052,906 B1 describes a synthetic transmembrane component).

[0216] The transmembrane domain can be derived from CD28, CD8a or TYRP-1, which confer superior receptor stability.

[0217] In one embodiment, the transmembrane domain is derived from CD8a. SEQ ID NO: 11: CD8a transmembrane domain IYIWAPLAGTCGVLLLSLVIT

[0218] In another embodiment, the transmembrane domain is derived from TYRP-1. SEQ ID NO: 12: TYRP-1 transmembrane domain IIAIAVVGALLLVALIFGTASYLI

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

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

[0221] In another embodiment, at least two costimulatory endodomains are used together with CD3ζ. In a specific embodiment, the two costimulatory 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 from OX40 and CD3ζ, endodomains from 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 the CAR with an activating endodomain may comprise the sequences set forth as SEQ ID NOs: 13-18, or variants thereof having at least 80% sequence identity. [ka] [ka]

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

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

[0225] Bispecific T cell-inducing molecules are a class of bispecific antibody-type molecules primarily developed for use as anticancer agents. Bispecific T cell-inducing molecules direct the host's 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, while the other binds to target cells such as tumor cells (via a tumor-specific molecule). Because bispecific molecules bind to both target cells and T cells, they bring the target cells into close proximity with the T cells, allowing the T cells to exert their effects, such as cytotoxic effects against cancer cells. Formation of the T cell:bispecific Ab:cancer cell complex induces signaling in the T cell, resulting in, for example, the release of cytotoxic mediators. Ideally, the agent only induces the desired signaling in the presence of target cells, resulting in selective killing.

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

[0227] 6.3. Bispecific T cell inducers The present invention also contemplates bispecific molecules that can selectively recognize TRBC1 and attract and activate T cells. For example, the molecule can be a BiTE.

[0228] Therefore, in another aspect, the present invention provides a bispecific T cell inducer (BiTE) comprising a TRBC1-specific antibody of the present invention or a 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" have been described in detail in the previous aspects, and their features and embodiments apply equally to this aspect of the present invention.

[0230] As used herein, the term "T cell activation domain" refers to a second domain that can activate T cells. The T cell activation domain can 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 comprise a signal peptide to aid in its production, which may cause it to be secreted by the host cell so that it can be harvested from the host cell supernatant.

[0232] The signal peptide may be at the amino terminus of the molecule. Bispecific molecules may have the general formula: signal peptide-variant antigen binding domain of the invention-T cell activation domain.

[0233] The bispecific molecule may comprise a spacer sequence connecting the variant antigen-binding domain and the T cell activation domain of the invention and spatially separating the two domains.

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

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

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

[0237] In another aspect, the present invention also provides nucleic acid sequences encoding the BiTEs 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" have been described in detail in connection with the above 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] It will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, it will be understood that, to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed, those skilled in the art can, using routine techniques, make nucleotide substitutions 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 the sequence that encode the same or similar amino acid sequences to avoid homologous recombination.

[0242] Nucleic acids according to the present invention may comprise DNA or RNA. Nucleic acids according to the present invention may be single-stranded or double-stranded. Nucleic acids according to the present invention may also be polynucleotides containing synthetic or modified nucleotides. Many 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. It should be understood that for the uses described herein, polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0243] The terms "variant," "homologue," or "derivative" in reference to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acid(s) from or to the sequence.

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

[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 encoding them, have been described in detail in connection with the above aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

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

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

[0248] 6.6.Cells Another aspect of the invention pertains to cells comprising the CAR of the invention.

[0249] The cell may contain a nucleic acid or vector of the invention.

[0250] The terms "CAR of the invention", "nucleic acid of the invention", and "vector of the invention" have been described in detail in connection with the above aspects of the invention, and their features and embodiments apply equally to these aspects of the invention.

[0251] The cell can be a cytolytic immune cell such as a T cell or an NK cell.

[0252] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated 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 the cell surface. There are various types of T cells, as summarized below.

[0253] T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or THF, which secrete different cytokines to promote different types of immune responses.

[0254] Cytolytic T cells (TC cells or CTLs) destroy virus-infected 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 long-term after an infection has resolved. Memory T cells rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells 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+. Memory T cells typically 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 primary role is to shut down T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

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

[0258] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are involved in interactions 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 interfere with the development of regulatory T cells, leading to the fatal autoimmune disease IPEX.

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

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

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

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

[0263] Cells according to this aspect of the invention can be generated ex vivo in the context of hematopoietic stem cell transplantation 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).

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

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

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

[0267] The cells of the invention can be made by a method comprising transducing or transfecting a cell with a vector of the invention comprising a nucleic acid sequence encoding a CAR.

[0268] The method for producing the cells of the present invention may further comprise the step of isolating the cells from a cell-containing sample derived from a subject or from another source listed above prior to the transduction or transfection step. If the cells are cytolytic cells, the sample is a cytolytic cell-containing sample derived from the subject.

[0269] The term "subject" or "individual" as used in the context of the present invention refers to a member of a mammalian species, preferably a human being, male or female, of any age or race.

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

[0271] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising a TRBC1-specific antibody of the present invention, or a TRBC2-specific antibody of the present invention, or a cell or cells of the present invention, or a 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" have been described in detail in connection with the above aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0273] The descriptions and embodiments of the pharmaceutical compositions of the above aspects of the invention apply equally to these aspects of the invention, and those skilled in the art will immediately know what modifications may be necessary to adapt the pharmaceutical compositions of the above aspects of the invention to this aspect.

[0274] 6.8. Treatment Method In another aspect, the present invention provides 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 a BiTE of the present invention for use in medicine.

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

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

[0277] This aspect of the invention may alternatively be formulated as the use of a TRBC1-specific antibody of the invention, or a TRBC2-specific antibody of the invention, or a cell or cells of the invention, or a BiTE of the invention in the manufacture of a medicament 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" have been described in detail in connection with the above aspects of the present invention, and their features and embodiments apply equally to these aspects of the present invention.

[0279] Methods for treating T-cell lymphoma and / or leukemia relate to therapeutic uses of the antibody conjugates of the invention that may be administered to a subject with an existing T-cell lymphoma and / or leukemia to alleviate, reduce or ameliorate at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0280] The descriptions and embodiments of the methods of treatment of the above aspects of the invention apply equally to these aspects of the invention, and those skilled in the art will immediately know what modifications may be necessary to adapt the methods of treatment of the above aspects of the invention to this aspect.

[0281] Diagnostic Agents TRBC1 + Against TRBC2 + It has previously been determined that the percentage of T cells from healthy donors expressing TRBC1 is 35% vs. 65%, i.e., the median percentage of total T cells expressing TRBC1 is 35% (range, 25-47%) (Maciocia et al., 2017, Nat Med, 23:1416-23). ​​Because T-cell lymphoma or leukemia is a clonal cancer (Maciocia et al., 2017; supra), the deregulated proliferation of malignant T cells characteristic of T-cell lymphoma or leukemia is due to a significantly skewed TRBC1 expression. + or TRBC2 + T cells (i.e., TRBC2 - or TRBC1 - Thus, by specifically binding to TRBC1 and therefore being able to distinguish between TRBC1 and TRBC2, the antibodies of the present invention constitute useful agents 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 of the present invention 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" have been described in detail in connection with the above aspect of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0284] The TRBC1-specific antibodies or TRBC2-specific antibodies of the present invention to be used in these assays may be labeled or unlabeled. As used herein, the term "detectable label" or "labeling agent" refers to a molecular label that allows for the detection, localization, and / or identification of the molecule to which it is bound using appropriate procedures and equipment for detection, for example, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Labeling agents suitable 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 require detection with additional reagents, such as labeled secondary antibodies. This is particularly useful for increasing the sensitivity of the detection method, as it allows the signal to be amplified. There are a wide range of conventional assays that can be used in the present invention using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), including Western blotting or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, flow cytometry or multiplexed detection techniques based on the use of protein microspheres, biochips, or microarrays containing the antibodies of the present invention. Other methods for detecting and quantifying TRBC1 using the mutant antigen-binding domains or antibodies of the present invention include affinity chromatography techniques or ligand binding assays.

[0285] The diagnostic agent may be used to diagnose T-cell lymphoma or leukemia.

[0286] The T-cell lymphoma or leukemia may be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0287] 6.10. Diagnostic Methods In another aspect, the present invention provides a method for diagnosing T-cell lymphoma or leukemia in a subject, the method comprising contacting a TRBC1-specific antibody of the present invention or a TRBC2-specific antibody of the present invention with a sample containing T cells from the subject.

[0288] The terms "TRBC1-specific antibody of the present invention" and "TRBC2-specific antibody of the present invention," "subject," and "sample" have been described in detail in connection with the above aspect 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 comprise a step of determining the total number of TRBC1-positive T cells in the sample. The diagnostic method of the present invention may further comprise a step of determining the total number of TRBC2-positive T cells in the sample. The diagnostic method of the present invention may further comprise a step of determining the total number of TRBC1-positive T cells and TRBC2-positive T cells in the sample.

[0290] The diagnostic methods of the present invention may further comprise the step of determining the total number of T cells in the sample, which may use 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 comprise a step of determining the proportion of TRBC1-positive T cells in the sample.

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

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

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

[0295] Those skilled in the art can use many conventional methods for detecting TRBC1 or TRBC2 in a sample, which 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 can be particularly useful for carrying out the diagnostic method of the present invention. The characteristics and specific embodiments of the diagnostic agent of the present invention have been defined above and apply equally to the diagnostic method of the present invention.

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

[0297] As understood by those skilled in the art, although 100% accuracy is preferred, prediction does not need to be accurate for 100% of the subjects to be diagnosed or evaluated.However, this term requires that a statistically significant portion of subjects can be identified as having an increased probability of having a given outcome.Whether the data obtained from a subject is statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, cross-validated classification rate, etc.Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.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 the 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 range of techniques and processes used to create images of the body (or its parts and functions), e.g., the human body, for clinical purposes such as medical treatments aimed at revealing, diagnosing, or examining diseases.

[0299] For this purpose, the mutant antigen-binding domains or antibodies of the present invention can be labeled by a suitable method known in the art, for example, by conjugation and / or loading with a suitable molecule, such as a radioisotope or fluorescent dye, to provide them as agents for imaging diagnostics such as radioimmunodiagnostics, positron emission tomography (PET), and endoscopic immunofluorescence. The mutant antigen-binding domains and antibodies of the present invention can be conjugated to a gamma-ray radioisotope 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 can be conjugated to a positron emitter and used in PET. The mutant antigen-binding domains and antibodies of the present invention can be conjugated to a fluorescent dye 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 above can be administered to an individual at an appropriate dose by any suitable route, for example, intravenously, and the location of TRBC1-positive T cells can be detected, determined, or measured by processes well known in the art. The methods and techniques used herein, including diagnostic imaging, are known to those skilled in the art, who will also be able to provide appropriate dosage formulations.

[0300] The T-cell lymphoma or leukemia to be diagnosed may be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

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

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

[0303] Thus, in another aspect, the present invention provides a method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, comprising: i) determining whether malignant T cells in a sample isolated from the subject express TRBC1 or TRBC2; ii) selecting cells, TRBC1-specific antibodies, TRBC2-specific antibodies, or BiTEs 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, hereinafter referred to as "first method of personalized medicine of the present invention," comprising:

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

[0305] The terms "cells of the present invention," "TRBC1-specific antibodies of the present invention," "TRBC2-specific antibodies of the present invention," "BiTEs of the present invention," "subjects," and "samples containing T cells" have been described in detail in connection with the above aspects of the present invention, and their features and embodiments apply equally to this aspect of the present invention.

[0306] The descriptions and embodiments of the personalized medicine method of the above aspect of the invention apply equally to this aspect of the invention, and those skilled in the art will immediately know what modifications may be necessary to adapt the personalized medicine method of the above aspect of the invention to this aspect. [Example]

[0307] Example [Example 1] Example 1: Antibody internalization of hJOVI-1 and KFN antibodies We tested the cellular uptake of the anti-TRBC1 antibody hJOVI-1 and the anti-TRBC2 antibody KFN in HPB-ALL cells engineered to express only TRBC1, only TRBC2, or knockout (KO) TRBCs. To this end, hJOVI-1 and KFN were conjugated in IgG format using Zenon pHrodo iFL red (Thermo Scientific), a pH-sensitive fluorophore with a 560 / 585 nm spectrum, according to the manufacturer's recommendations. An irrelevant 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 for 24 hours at 37°C and 5% CO2. 5 x 10 cells were added in 100 μl of RPMI 10% FBS. 4Test antibodies hJOVI-1, KFN, and anti-HEL were applied at 5 μg / ml in 96-well plates with cells / well. After 24 hours, cells were washed, harvested, and stained with 1:100 LIVE / DEAD™ Fixable Violet Dead Cell Stain (Thermofisher) for 10 minutes before analysis by flow cytometry using a Fortessa flow cytometer (BD).

[0308] The results shown in Figure 4 demonstrate that both anti-TRBC antibodies, hJOVI-1 and KFN, internalized well into HPB-ALL cells. However, the KFN antibody, which binds to its target at the same epitope but with lower affinity, showed improved internalization compared with the higher-affinity hJOVI-1. The specificity of hJOVI-1 and KFN was maintained as antibody conjugates, as hJOVI-1 internalized only into HPB-ALL TRBC1 cells and KFN internalized only into HPB-ALL TRBC2 cells.

[0309] [Example 2] Example 2: TRBC antibody affinity and binding kinetics Surface plasmon resonance (SPR) was performed on several of the hJOVI-1 antibody variants to confirm their affinity, association, and dissociation rates. Detailed sequences of these variants, Mut1 to Mut15, used in the Examples section of this patent application, are shown in Tables 1 and 2. These anti-TRBC antibody variants have specificity for 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 binding kinetics determination, mouse anti-human IgG (GE Healthcare) was covalently coupled to a CM5 Sensor Chip (GE Healthcare). Anti-TRBC1 or anti-TRBC2 antibodies were captured on the flow cell, and various concentrations of the interacting partner protein (TRBC1 or TRBC2) were injected over the flow cell at a flow rate of 30 ml / min at a temperature of 25 °C. Double reference subtraction was performed using buffer alone. Kinetic rate constants were obtained by curve fitting according to a 1:1 Langmuir binding model.

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

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

[0313] [Example 3] Example 3: Antibody internalization of anti-TRBC1 antibody The internalization of the anti-TRBC1 antibody selected in Example 2 (FIG. 6) was evaluated in HPB-ALL cells (TRBC1, TRBC2, and KO). To this end, the anti-TRBC1 antibody was conjugated in IgG format using the pH-sensitive fluorophore Zenon pHrodo iFL Green Reagent (Thermo Scientific), which has a 509 / 533 nm spectrum, according to the manufacturer's recommendations. An irrelevant anti-HEL antibody was also conjugated for use as a control. The conjugated antibody was incubated with HPB-ALL TRBC1, HPB-ALL TRBC2, and HPB-ALL KO cells for 9 hours at 37°C and 5% CO2. 5x10 IgG antibodies were added in 100 μl of RPMI 10% FBS. 4 Test antibodies were applied at 5 μg / ml in 96-well plates with cells / well. After 9 hours, cells were washed, harvested, and stained with 1:1000 of the fixable viability dye eFluor™ 780 (eBioscience) for 10 minutes before analysis by flow cytometry using a Fortessa flow cytometer (BD).

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

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

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

[0317] Internalization experiments using Mut11 and Mut15 were performed as described in Example 3.

[0318] The results showed that both Mut11 and Mut15 were well internalized (Figure 8). Furthermore, the results demonstrated that the internalization obtained with these two antibodies was much improved compared to that of hJovi1. Mut11 mutant showed the greatest internalization in TRBC1 cells while maintaining antigen specificity. Similarly, KFN mutant Mut15 showed optimal internalization and specificity for TRBC2 cells.

[0319] [Example 5] Example 5: Generation of TRBC-specific ADCs To test the efficacy of the TRBC1 and TRBC2 ADC molecules, Mut11 and Mut15 antibodies were conjugated to monomethyl auristatin E (MMAE). Anti-HEL was used as a control. Briefly, the antibodies were generated and highly purified. Antibody-drug conjugates were generated using the linker mc-vc-PAB-MMAE (Figure 9A). Briefly, a reducing agent was first used to liberate the nucleophilic cysteine ​​residue from the interchain disulfide bond of the reduced antibody. The reduced cysteine ​​was then conjugated with the drug-linker conjugate. Conjugation conditions were optimized to identify ADCs with the desired drug-to-antibody ratio (DAR). The drug-to-antibody ratio was assessed by hydrophobic interaction chromatography on a TSKgel Butyl-NPR column (2.5 μm, 4.6 × 100 mm) and was estimated to be 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 ability of the antibodies to be internalized (Fig. 9B).

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

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

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

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

[0325] [Example 8] Example 8: Functional characterization of anti-TRBC1 CAR: cytokine production To assess the functional capacity of anti-TRBC1 mutant CAR-T cells against TRBC1, we used a plate-binding assay to immobilize TRBC1 or TRBC2 before adding CAR-T cells. After 72 hours, culture supernatants were collected and IFN-γ production was measured by ELISA.

[0326] [Example 9] Example 9: Functional characterization of anti-TRBC1 CAR: Cytotoxicity assay To determine the ability of the anti-TRBC1 triple mutant to target TRBC1, we set up a cytotoxicity assay 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 were cultured at a 1:1 (E:T) ratio with either of the following cells. Target cell recovery was measured by flow cytometry after 72 hours of culture and used to establish the cytotoxicity of the CAR-T cells.

[0327] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be 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 a TCR beta chain constant region (TRBC), said antibody being capable of binding to a TCR beta chain constant region (TRBC) in 0.001 seconds. -1 ~0.3 seconds -1 The dissociation rate constant (k d ) an antibody conjugate having (Item 2) The antibody is -1 ~0.1 seconds -1 k in the range d 2. The antibody conjugate of item 1, having the following structure: (Item 3) 3. The antibody conjugate of any of items 1 or 2, wherein the antibody is conjugated to a chemotherapeutic entity, a radionuclide or a detectable entity. (Item 4) 4. The antibody conjugate of item 3, wherein the chemotherapeutic entity is a tubulin inhibitor. (Item 5) 5. The antibody conjugate of item 4, wherein the tubulin inhibitor is MMAE. (Item 6) 6. The antibody conjugate according to any of items 1 to 5, wherein the antibody conjugate has increased internalization upon binding to target cells compared to the internalization of a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2. (Item 7) 7. The antibody conjugate according to any one of items 1 to 6, wherein the antibody specifically binds to TRBC1. (Item 8) The antibody has the following mutations or combinations of mutations compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth 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; or - Y102M in the VH domain 8. The antibody conjugate according to item 7, comprising one of: (Item 9) The antibody has the following mutations compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2: G106A in the VH domain 9. The antibody conjugate of item 8, comprising: (Item 10) 7. The antibody conjugate according to any one of items 1 to 6, wherein the antibody specifically binds to TRBC2. (Item 11) The antibody has the following mutations or combinations of mutations compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth 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 11. The antibody conjugate according to item 10, comprising one of: (Item 12) The antibody has the following combination of mutations compared to a reference antibody having a VH domain having the sequence set forth in SEQ ID NO: 1 and a VL domain having the sequence set forth in SEQ ID NO: 2: - T28K, Y32F, A100N and N103L in the VH domain and N35R in the VL domain 12. The antibody conjugate according to item 11, comprising one of: (Item 13) 13. The antibody conjugate of any one of items 1 to 12 for use in the treatment of T-cell lymphoma or leukemia. (Item 14) 14. The antibody conjugate for use according to Item 13, wherein the treatment of T-cell lymphoma or leukemia in a subject comprises administering the antibody conjugate to the subject to selectively deplete malignant T cells along with normal T cells that express the same TRBCs as the malignant T cells, but not normal T cells that express TRBCs that are not expressed by the malignant T cells. (Item 15) The antibody conjugate for use described in Item 14, wherein the method further comprises examining the TCR β chain constant region (TCRB) of malignant T cells from the subject to determine whether the malignant T cells from the subject express TRBC1 or TRBC2. (Item 16) 16. The antibody conjugate for use according to any of items 13 to 15, wherein the T-cell lymphoma or leukemia is selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia and T-cell acute lymphoblastic leukemia. (Item 17) 13. The antibody conjugate according to any one of items 3 to 12, for use in a method for targeting the delivery of a chemotherapeutic agent to cells expressing TRBCs in a subject. (Item 18) 13. A pharmaceutical composition comprising the antibody conjugate according to any one of items 1 to 12 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. (Item 19) 1. A method for selecting an appropriate therapy for treating a subject suffering from T-cell lymphoma or leukemia, comprising: i) determining whether malignant T cells in a sample isolated from the subject express TRBC1 or TRBC2; ii) selecting an antibody conjugate for use according to any one of items 13 to 16 based on the TRBC1 or TRBC2 expression of the malignant T cells; A method comprising: (Item 20) 17. A method for selecting a subject suffering from T-cell lymphoma or leukemia to receive a therapy comprising the antibody conjugate for use according to any of items 13 to 16, comprising: i) determining whether malignant T cells in a sample isolated from the subject express TRBC1 or TRBC2; ii) selecting the subject for a therapy based on the antibody conjugate for use according to any one of items 13 to 16 based on the TRBC1 or TRBC2 expression of the malignant T cells; A method comprising:

Claims

[Claim 1] The invention described in the specification.

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