Antibody compositions for reducing cross-reactivity

Abstract

The invention relates to antibody compositions for reducing cross reactivity between antibodies specific to related antigens, methods using such compositions and kits comprising such compositions.

Description

[0001] ANTIBODY COMPOSITIONS FOR REDUCING CROSS-REACTIVITY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to antibody compositions for reducing cross reactivity between antibodies specific to related antigens, methods using such compositions and kits comprising such compositions.

[0004] Background of the invention

[0005] Each T cell expresses a TCR (T Cell Receptor) which is a molecular complex made of two units: a recognition unit composed of either ap or y<5 heterodimers, which are present on the cell surface in a mutually exclusive manner, and a transducing unit, the CD3 complex, common to ap and y<5 heterodimers, which triggers the T cell when the recognition unit is engaged with the antigen. The recognition units recognize a large variety of antigens thanks to a somatic genomic recombination of the four TCR gene loci (a, p, y and 5) which takes place in the thymus (T-cell ontogeny). Each locus is composed of several V (Variable) segments, very short D segments (for p and 5 loci only), and short J segments, and one or two C (Constant) genes.

[0006] TCRap T-cell precursors undergo rearrangement of the TCR genes in the thymus, including:

[0007] • Random selection of one of 64 TCR variable p chains

[0008] • Random selection of one of two mutually exclusive TCR constant p chains (TRBC1 and TRBC2).

[0009] The TCR produced from this rearrangement is expressed on the surface of all normal mature T cells. Therefore, normal T-cell populations include clonal populations that will be either TRBC1+ or TRBC2+, as a result of the selection of one of the two mutually exclusive TCR constant p chains.

[0010] Commonly, neoplastic T-cells are TRBC-restricted, i.e. make up a clonal population of T-cells. However, identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays.

[0011] Recently, the T-cell Receptor Constant p Chains-1 and 2 (TRBC1 and TRBC2), mutually expressed at the surface of all mature TCRap T-cells, have emerged as a potential low-cost, rapid, and specific T-cell clonality test by flow cytometry.

[0012] The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) p- chain constant regions (TRBC) provides an opportunity to facilitate the detection of clonal TCRap+ T cells based on TRBC-restriction. Anti-TRBC1 and anti-TRBC2 antibodies are specific to epitopes located in the constant domain of the T Cell Receptor (TCR) Chain (Figure 1A), hence, those antibodies are particularly useful to identify clonality among T cells expressing either TRBC1 or TRBC2 (Figure 1 B).

[0013] However, differences between TRBC1 and TRBC2 epitopes are small (only 4 different amino acids, see Figure 2). Consequently, a non-specific reactivity exists between those two antibodies, i.e. an anti-TRBC1 antibody can react partially toward TRBC2 epitope and reversely (see Figure 3; arrows), which cannot be overcome by optimization of the respective antibodies alone. The problem of cross-reactivity generally arises between closely related variants of a gene or protein (e.g. homologs).

[0014] Hence, ways of eliminating non-specific binding of an antibody to epitopes that are closely related to its binding epitope are urgently needed. Reagents and methods to help identify clonal populations of cells that express proteins with closely related epitopes are needed.

[0015] Objectives and Summary of the Invention

[0016] The inventors have surprisingly found that they could reduce non-specific binding of an antibody that is specific for an epitope and exhibits non-specific binding with a closely related epitope, without affecting the specific binding of the same antibody. This situation can occur when two or more closely related variants of a protein exists, caused, for example, by gene duplication events throughout evolutionary history of the species.

[0017] To this end, they used a purified, unconjugated second antibody specific for the non-target epitope to block binding to the non-target epitope. The reagent and methods comprising one conjugated and one non-conjugated antibody require less process steps and materials, since only one signal needs to be processed.

[0018] Hence, in first aspect, the invention provides a composition comprising a first antibody or an epitopebinding fragment thereof specific for a first variant of a protein of interest (POI), wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and a second antibody or an epitope-binding fragment thereof specific for a second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0019] The composition reduces cross-reactivity of the first antibody or an epitope-binding fragment thereof compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0020] Conversely, the composition enhances specific binding of the first antibody or an epitope-binding fragment thereof to the first variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0021] Furthermore, the composition reduces non-specific binding of the first antibody or an epitope-binding fragment thereof to the second variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0022] Providing a composition with only one conjugated antibody or epitope-binding fragment thereof reduces the need for signal processing and reduces time and reagent cost. Using two or more conjugated antibodies necessitates finding matching tags or chromophores when planning experiments, purchasing additional reagents, doing additional staining and washing steps in the process, and read-out of several signals, which results in additional analysis.

[0023] In one embodiment, the detectable moiety is a chromogenic species or a fluorescent species.

[0024] The molar ratio of the first and second antibody may need to be optimised for any pair of antibodies and targets. To achieve good results, the difference between the amounts of each antibody should not be more than 10-fold. Hence, in one embodiment, the molar ratio between the first antibody or an epitope-binding fragment thereof and the second antibody or an epitope-binding fragment thereof is between 1 :0.1 and 1 :10. In a preferred embodiment, the molar ratio between the first antibody or an epitope-binding fragment thereof and the second antibody or an epitope-binding fragment thereof is between 1 :0.25 and 1 :2. Good results can be achieved with a molar ratio of 1 :1 ± 0.25, hence this ratio is especially preferred.

[0025] In one embodiment, the antibody concentration of each antibody or epitope-binding fragment thereof is between 0.1 - 2pg / 1 OOpI per sample. In one embodiment, the concentration of each antibody or epitope-binding fragment thereof is between 0.25 - 1 ,5pg / 1 OOpI per sample. Preferably, the antibody concentration of each antibody or epitope-binding fragment thereof is between 0.4 - 0.6pg / 1 OOpI per sample.

[0026] In some embodiments, the composition further comprises an excipient or a stabilizer.

[0027] Another aspect of the invention is the use of the composition described herein in the detection of the first variant of the POI.

[0028] In another aspect, the invention relates to a kit comprising a first antibody or an epitope-binding fragment thereof conjugated with a detectable moiety and a second antibody or an epitope-binding fragment thereof not conjugated with a detectable moiety.

[0029] In one embodiment, the first and second antibody or epitope-binding fragment thereof are provided in separate vials. In another embodiment, the first and second antibody or epitope-binding fragment thereof are provided in one vial.

[0030] In another aspect, the invention relates to a kit comprising a first composition comprising a first antibody or an epitope-binding fragment thereof conjugated with a detectable moiety and a second antibody or an epitope-binding fragment thereof not conjugated with a detectable moiety; and further comprising a second composition comprising the second antibody or an epitope-binding fragment thereof conjugated with a detectable moiety and the first antibody or an epitope-binding fragment thereof not conjugated with a detectable moiety.

[0031] In one embodiment, the first composition and the second composition are comprised in separate vials.

[0032] In another aspect, the invention also provides an in vitro method of enhancing the specificity of a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI) in a cell population expressing first variant and a further second variant of the POI in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising a first antibody or an epitope-binding fragment thereof specific for the first variant of the POI, wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety, and a second antibody or an epitope-binding fragment thereof specific for second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each antibody to their respective target epitope; and c) detecting the conjugated first antibody or an epitope-binding fragment thereof.

[0033] The method of the invention is particularly suitable for POI that are closely related on an amino acid sequence level. In one embodiment, the first and second variants of the POI are homologs, paralogs, orthologs, isoforms or mutants.

[0034] In some embodiments, the amino acid sequence of first variant of the POI and the amino acid sequence of second variant of the POI are at least 90% identical, the amino acid sequence of first variant of the POI and the amino acid sequence of second variant of the POI are at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical.

[0035] The close amino acid sequence similarity of the variants of the POI usually results in epitopes that are at least partially overlapping, or even completely overlapping, with only small amino acid sequence differences. The first antibody or an epitope-binding fragment thereof specifically binds to a first epitope in the first variant of the POI and the second antibody or an epitope-binding fragment thereof specifically binds to a second epitope in the second variant of the POI . In one embodiment, the amino acid sequence of the first epitope and the amino acid sequence of the second epitope are at least 50% identical.

[0036] In one embodiment, the amino acid sequence of the first epitope and the amino acid sequence of the second epitope are at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical.

[0037] Likewise, in one embodiment, the first epitope and the second epitope are in the same location within the amino acid sequence of the first and second variant of the POI.

[0038] Essentially, the second antibody or an epitope-binding fragment thereof binds to and blocks the second epitope in the second variant of the POI, which is therefore not available for non-specific binding by the first antibody or an epitope-binding fragment thereof. Hence, in one embodiment, the binding of the second antibody or an epitope-binding fragment thereof prevents binding of the first antibody or an epitope-binding fragment thereof to epitopes recognised by the first antibody or an epitope-binding fragment thereof comprised in the second variant.

[0039] A particularly interesting application ofthe composition and the methods described herein is for the detection of a clonal subpopulation of T-cells. T-cells express either TRBC1 or TRBC2 in a mutually exclusive manner. Interestingly, the two p constant chain domains TRBC1 and TRBC2 only differ in 4 amino acids (N4K, K5N, F36Y, V135E, see for example Maciocia et al. Nat Med. 2017, 23(12):1416- 1423.) The binding epitope of specific anti-TRBC1 and specific anti-TRBC2 antibodies can therefore only be in locations where the amino acid sequence differs. Thus, in some embodiments, the binding epitopes comprise N4 / K4 and K5 / N5 (numbering of TRBC1 and TRBC2, respectively).

[0040] Hence, in one embodiment, the first antibody is anti-TRBC1 or an epitope-binding fragment thereof and the second antibody is anti-TRBC2 or an epitope-binding fragment thereof. In particular, the anti-TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0041] Hence, in one embodiment, the composition comprises an anti-TRBC1 antibody or an epitopebinding fragment thereof comprising a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6; wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated to a chromophore or a fluorophore, and an unconjugated anti-TRBC2 antibody or an epitope-binding fragment thereof.

[0042] In one embodiment, the anti-TRBC2 antibody or an epitope-binding fragment thereof comprises the same CDR sequences as the anti-TRBC1 antibody but comprises the amino acid substitutions corresponding to T28K, Y32F, A96N and N99M, according to KABAT numbering. These amino acid substitutions correspond to T28K, Y32F, A100N and N103M of the sequence of mouse JOVI.1 depicted in SEQ ID NO: 17.

[0043] In one embodiment, the anti-TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, and a CDR3 sequence according to SEQ ID NO: 8.

[0044] In one embodiment, the anti-TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0045] In another embodiment, the first antibody is anti-TRBC2 or an epitope-binding fragment thereof and the second antibody is anti-TRBC1 or an epitope-binding fragment thereof.

[0046] In particular, the anti-TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0047] Hence, in one embodiment, the composition comprises an anti-TRBC2 antibody or an epitopebinding fragment thereof comprising a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6; wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated to a chromophore or a fluorophore, and an unconjugated anti-TRBC1 antibody or an epitope-binding fragment thereof.

[0048] In one embodiment, the anti-TRBC2 antibody or an epitope-binding fragment thereof comprises the same CDR sequences as the anti-TRBC1 antibody but comprises the amino acid substitutions corresponding to K28T, F32Y, N96A and M99N, according to KABAT numbering. These amino acid substitutions correspond to K28T, F32Y, N100A and M103N according to the numbering of full-length sequence of mouse anti-TRBC2 depicted in SEQ ID NO: 19. In one embodiment, the anti-TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , and a CDR3 sequence according to SEQ ID NO: 3.

[0049] In one embodiment, the anti-TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0050] In another aspect, the invention relates to a kit comprising an anti-TRBC1 antibody or an epitopebinding fragment thereof conjugated with a detectable moiety and an anti-TRBC2 antibody or an epitope-binding fragment thereof not conjugated with a detectable moiety.

[0051] In one embodiment, the anti-TRBC1 and anti-TRBC2 antibodies or epitope-binding fragments thereof are provided in separate vials. In another embodiment, the anti-TRBC1 and anti-TRBC2 antibodies or epitope-binding fragments thereof are provided in one vial.

[0052] In another aspect, the invention relates to a kit comprising an anti-TRBC2 antibody or an epitopebinding fragment thereof conjugated with a detectable moiety and an anti-TRBC1 antibody or an epitope-binding fragment thereof not conjugated with a detectable moiety.

[0053] In one embodiment, the anti-TRBC2 and anti-TRBC1 antibodies or epitope-binding fragments thereof are provided in separate vials. In another embodiment, the anti-TRBC2 and anti-TRBC1 antibodies or epitope-binding fragments thereof are provided in one vial.

[0054] In another aspect, the invention also provides a kit comprising a first composition comprising an anti- TRBC1 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti-TRBC2 antibody or a epitope-binding fragment thereof not conjugated with a detectable moiety; and further comprising a second composition comprising an anti-TRBC2 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti-TRBC1 antibody or a epitopebinding fragment thereof not conjugated with a detectable moiety.

[0055] In one embodiment, the first composition and the second composition are comprised in separate vials.

[0056] Figure Legends

[0057] Figure 1 : Schematic overview of anti-TRBC1 and anti-TRBC2 antibodies. A: Anti-TRBC1 and anti-TRBC2 antibodies are specific to epitopes located in the constant domain of the T Cell Receptor (TCR) p Chain. B: The expression of TRBC1 and TRBC2 is mutually exclusive: a T cell expresses TRBC1 or TRBC2. Those antibodies are particularly useful to identify clonality among T cells expressing either TRBC1 or TRBC2. Figure adapted from Horna, P., et al. Dual T-cell constant p chain TRBC1 and TRBC2 staining for the identification of T -cell neoplasms by flow cytometry. Blood Cancer J. 14, 34 (2024).

[0058] Figure 2: Schematic overview of epitope recognition of anti-TRBC1 (JOVI.1) and anti-TRBC2 antibodies on TCR (T Cell Receptor). Highlighted amino acids indicate sequence differences in TRBC1 and TRBC2 p Chain and sequence differences in JOVI.1 and Anti-TRBC2 CDR1 and CDR3. Figure adapted from Horna, P., et al. Dual T-cell constant p chain TRBC1 and TRBC2 staining for the identification of T-cell neoplasms by flow cytometry. Blood Cancer J. 14, 34 (2024).

[0059] Figure 3: Double staining of Lymphocytescells with anti-TRBC1-PE and anti-TRBC2-APC conjugates. Flow cytometry plot showing anti-TRBC1-PE intensity (X axis) and anti-TRBC2-APC intensity (Y axis). Arrows highlight areas of non-specific reactivity between anti-TRBC1 antibody and TRBC2 epitope and anti-TRBC2 antibody and TRBC1 epitope. PE: Phycoerythrin; APC: Allophycocyanin; LY: Lymphocytes.

[0060] Figure 4: Single staining of Lymphocytes with anti-TRBC1-PE conjugate. Flow cytometry plots showing anti-TRBC1-PE intensity (X axis) and side scatter intensity (SS INT, Y axis). A: Formulation of anti-TRBC1-PE conjugate without any additional antibody. B: Formulation of anti-TRBC1-PE conjugate with unconjugated, purified anti-TRBC2 antibody. PE: Phycoerythrin.

[0061] Figure 5: Single staining of Lymphocytes with anti-TRBC2-APC conjugate. Flow cytometry plots showing anti-TRBC2-APC intensity (X axis) and side scatter intensity (SS INT, Y axis). A: Formulation of anti-TRBC2-APC conjugate without any additional antibody. B: Formulation of anti- TRBC2-APC conjugate together with unconjugated, purified anti-TRBC1 antibody. APC: Allophycocyanin.

[0062] Figure 6: Histograms of flow cytometry data from cell lines DAUDI (no TRBC), JURKAT (TRBC1) and HPB-ALL (TRBC2). A: Isotypic control (lgG2a-PE) on all cell types. B: Anti-TRBC1-PE alone on all cell types. C: Anti-TRBC1-PE and purified, unconjugated anti-TRBC2. Arrow indicates shift in signal in TRBC1 -negative HPB-ALL cell line. RMFI: relative mean fluorescence intensity.

[0063] Figure 7: Histograms of flow cytometry data from cell lines DAUDI (no TRBC), JURKAT (TRBC1) and HPB-ALL (TRBC2). Overlay plot showing anti-TRBC1-PE staining and isotypic control (lgG2a-PE) staining. Top row shows anti-TRBC1-PE alone, bottom row shows anti-TRBC1-PE with anti-TRBC2 purified, unconjugated antibody.

[0064] Figure 8: Histograms of flow cytometry data from cell lines DAUDI (no TRBC), JURKAT (TRBC1) and HPB-ALL (TRBC2). A: Isotypic control (lgG1-APC) on all cell types. B: Anti-TRBC2- APC alone on all cell types. C: Anti-TRBC2-APC and purified, unconjugated anti-TRBC1. Arrow indicates shift in signal in TRBC2-negative JURKAT cell line.

[0065] Figure 9: Histograms of flow cytometry data from cell lines DAUDI (no TRBC), JURKAT (TRBC1) and HPB-ALL (TRBC2). Overlay plot showing anti-TRBC1-PE staining and isotypic control (lgG1-APC) staining. Top row shows anti-TRBC2-APC alone, bottom row shows anti-TRBC2-APC with anti-TRBC1 purified, unconjugated antibody.

[0066] DETAILED DESCRIPTION OF THE INVENTION

[0067] Definitions

[0068] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0069] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.

[0070] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0071] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a compound is defined to be obtainable from a specific source, this is also to be understood to disclose a compound which is obtained from this source.

[0072] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

[0073] Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

[0074] The term “epitope-binding antigen fragment” as used herein refers to amino acid sequences that comprise less than a full-length antibody amino acid sequence but retain full functionality of the antibody they are derived from. An antibody usually consist of two heavy chains and two light chains, wherein each of the light chains consist of a variable and a constant domain, and each of the heavy chains consist of a variable and three constant domains. The antigen-binding site is located in the variable region. Typical fragments of antibodies that retain epitope-binding capabilities are the Fab (fragment antigen-binding) region, F(ab') fragment, F(ab')2 fragment, F(ab)c fragment, single-chain variable fragment (scFv, a fusion protein of the variable regions of the heavy and light chains), and single-domain antibody (sdAb, or nanobody, a single monomeric heavy chain variable antibody domain). In one embodiment, the epitope-binding antigen fragment as used herein comprises CDR 1 , CDR 2 and CDR 3 of its respective antibody, i.e. the complementarity-determining regions of the heavy chain variable region. In one embodiment, the epitope-binding antigen fragment is selected from the group consisting of Fab fragment, F(ab') fragment, F(ab')2 fragment, F( ab )c fragment, single chain variable fragment (scFv) and single domain antibody fragment (sdAB).

[0075] The term “composition” as used herein refers to a mixture comprising effective amounts of the components. The composition may be liquid or soluble (e.g. a powder). In the present case, the composition comprises at least two antibodies and optionally further components such as buffers or other excipients.

[0076] A “protein of interest” as used herein refers to a marker protein or a therapeutically relevant protein of which at least two closely related variants occur. The variants may be either naturally occurring or synthetic. For example, the protein of interest may exist in a wild-type variant and a mutated variant that comprises amino acid substitutions. In other cases, there may be several homologs of the protein of interest. The term “variant(s) of a protein of interest” as used herein refers to two or more highly similar proteins that originate from a single gene or gene family, including splice variants (i.e. isoforms), homologs (having shared ancestral origin) or mutants. Homologs include orthologs (diverged after speciation event), or paralogs (diverged after an exon, gene or genome duplication event). For example, TRBC1 and TRBC2 are variants of TRBC. TRBC1 and TRBC2 are paralogs.

[0077] The term “TRBC” as used herein refers to the T-cell receptor beta chain constant region in general. In mouse and human, there are two variants of TRBC, called TRBC1 and TRBC2, that have different coding regions as shown in Figure 1 .

[0078] The terms “cross-reactivity” or “non-specific binding” as used herein refers to binding of an antibody or epitope-binding fragment thereof to an epitope that is not the intended target. Specifically, “nonspecific binding” refers to binding of a closely related epitope that is not the target epitope.

[0079] As used herein, the term “excipient”, “carrier” or “stabilizer” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).

[0080] As used herein, the terms “chromogenic species” refers to a colourless molecules consisting of a chromophore and a specific enzymatic substrate. The term “chromophore” refers to a molecule which absorbs light at a particular wavelength and emits colour.

[0081] The term “fluorescent species” or “flourophore” refers to a fluorescent chemical compound that absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. Examples of fluorophores useful as detectable moieties of the invention include, but are not limited to, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, 3- Hydroxyisonicotinaldehyde, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7, PE-Cy5.5, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, G-Dye100, G-Dye200, G- Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhoamine B, Texas Red, Snv428 (SuperNova V428), SuperNova v605, SuperNova v786, other polymer dyes, Krome Orange, ECD, Allophycocyanin (APC), APC tandems, A700, A750, and APC- Cy7 conjugates.

[0082] Compositions

[0083] The invention provides a composition comprising a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI), wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and a second antibody or an epitope-binding fragment thereof specific for a second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety. In one embodiment, the invention provides a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti- TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0084] In one embodiment, the invention provides a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti- TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0085] Hence, in one embodiment, the invention provides a composition comprising an anti-TRBC1 antibody or a epitope-binding fragment thereof and an anti-TRBC2 antibody or a epitope-binding fragment thereof, wherein only one of the antibodies is conjugated with a detectable moiety.

[0086] In one aspect, the invention also relates to the use of the compositions of the invention in the detection of TRBCI and / or TRBC2.

[0087] The invention further provides a kit comprising an anti-TRBC1 antibody or a epitope-binding fragment thereof and an anti-TRBC2 antibody or a epitope-binding fragment thereof, wherein only one of the antibodies is conjugated with a detectable moiety.

[0088] In one embodiment, the invention provides a kit comprising an anti-TRBC1 antibody or an epitopebinding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti- TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0089] In one embodiment, the invention provides a kit comprising an anti-TRBC2 antibody or an epitopebinding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti- TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0090] In one embodiment, the anti-TRBC1 antibody or a epitope-binding fragment thereof and anti-TRBC2 antibody or a epitope-binding fragment thereof are provided in separate vials. In another embodiment, the anti-TRBC1 antibody or a epitope-binding fragment thereof and anti-TRBC2 antibody or a epitope-binding fragment thereof are provided in the same vial.

[0091] The invention further provides a kit comprising a first composition comprising an anti-TRBC1 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti- TRBC2 antibody or a epitope-binding fragment thereof not conjugated with a detectable moiety; and further comprising a second composition comprising an anti-TRBC2 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti-TRBC1 antibody or a epitopebinding fragment thereof not conjugated with a detectable moiety.

[0092] In one embodiment, the first composition and the second composition are comprised in separate vials.

[0093] In another embodiment, each antibody is comprised in a separate vial.

[0094] In one embodiment, the detectable moiety is selected from the group consisting of Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, 3-Hydroxyisonicotinaldehyde, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7, PE-Cy5.5, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, G-Dye100, G-Dye200, G-Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhoamine B, Texas Red, Snv428 (SuperNova V428), SuperNova v605, SuperNova v786, other polymer dyes, Krome Orange, ECD, Allophycocyanin (APC), APC tandems, A700, A750, and APC-Cy7 conjugates.

[0095] In one particular aspect, the invention provides a kit comprising:

[0096] (i) a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; and

[0097] (ii) a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 8, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6.

[0098] In another aspect, the invention provides a kit comprising:

[0099] (i) a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; and

[0100] (ii) a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19.

[0101] In another aspect, the invention provides a kit comprising:

[0102] (i) a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; and

[0103] (ii) a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19; wherein the detectable moiety is selected from the group consisting of Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, 3-Hydroxyisonicotinaldehyde, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7, PE-Cy5.5, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, G-Dye100, G-Dye200, G-Dye300, G-Dye400, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhoamine B, Texas Red, Sn V428 (SuperNova V428), SuperNova v605, SuperNova v786, other polymer dyes, Krome Orange, ECD, Allophycocyanin (APC), APC tandems, A700, A750, and APC-Cy7 conjugates.

[0104] In another aspect, the invention provides a kit comprising:

[0105] (i) a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; and

[0106] (ii) a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19; wherein the detectable moiety of (i) comprises R- Phycoerythrin (PE) and the detectable moiety of (ii) comprises Allophycocyanin (APC).

[0107] In another aspect, the invention provides a kit comprising: a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19.

[0108] In another aspect, the invention provides a kit comprising: a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19.

[0109] In another aspect, the invention provides a kit comprising: a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19, wherein the detectable moiety is R-Phycoerythrin (PE).

[0110] In another aspect, the invention provides a kit comprising: a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19, wherein the detectable moiety is Allophycocyanin (APC).

[0111] In another aspect, the invention provides a kit comprising: a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2, wherein the anti-TRBC2 antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 , wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; wherein the anti-TRBC1 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 17; and wherein the anti-TRBC2 antibody or epitope-binding fragment thereof has the amino acid sequence depicted in SEQ ID NO: 19, wherein the detectable moiety is SNv428. Method

[0112] In one aspect, the invention provides an in vitro method of enhancing specificity of an anti-TRBC1 antibody in a cell population expressing TRBC1 and TRBC2 in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 conjugated with a detectable moiety and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2 not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each TRBC antibody to their respective target epitope; c) detecting the detectable moiety.

[0113] In another aspect, the invention provides an in vitro method of enhancing specificity of an anti- TRBC2 antibody in a cell population expressing TRBC1 and TRBC2 in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2 conjugated with a detectable moiety and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each TRBC antibody to their respective target epitope; c) detecting the detectable moiety.

[0114] The in vitro method according to the invention may be performed on any sample comprising TRCB1 and TRCB2. In one embodiment, the in vitro method is performed on whole blood samples or cell line samples.

[0115] In some embodiments, the method of the invention is performed with a further third antibody which is used to identify a T-cell subpopulation. T-cell subpopulations from whole blood samples are identified using the markers CD3, anti-TCRa / p, CD7, TCRg / d, CD4 or CD8. Hence, in some embodiments, the composition comprises i) conjugated anti-TRBC1 or a epitope-binding fragment thereof, unconjugated anti-TRBC2; or ii) conjugated anti-TRBC2 or a epitope-binding fragment thereof, unconjugated anti-TRBC1 ; and at least one further conjugated antibody selected from the group consisting of CD3, anti-TCRa / p, CD4,CD8 and anti-TRCg / d. These antibodies can be used to gate specifically a / b Tcell subpopulation

[0116] In one embodiment, the method is a flow cytometry method.

[0117] Flow cytometry involves preparing a liquid particle source, such as a medium comprising cells, which are then are hydrodynamically focused in a stream of fluid. An optical energy source, such as a laser, is directed toward the fluid stream, and one or more detectors are positioned to receive scattered light from the particles, light emitted from fluorescent tags on the particles, or both. The detectors may be positioned both inline with the optical energy source, and at various angles to the optical energy source. Different detectors may be sensitive to different wavelengths of optical energy. By analyzing the signals received from the detectors, information about the physical or chemical properties of the particles may be determined. Histograms and other graphical plots may be generated that may depict particle counts or concentrations of particles containing the fluorescent tags. The staining procedure using the compositions and methods of the invention comprises anti- TRBC1 and anti-TRBC2 antibodies, which target different surface markers, to identify TRBC1 positive and TRBC2 positive cells. After staining the sample is loaded into the flow cytometer, where the fluorescently labeled cells are excited as they pass by the laser emitting light at various wavelengths which are detected by the flow cytometer. Description of flow cytometry methods useful for the invention are commonly known and include for example, Pockley et al. Curr Protoc Toxicol . 2015 Nov 2:66:18.8.1-18.8.34.

[0118] Antibodies and fragments thereof

[0119] Anti-TRBC1 antibody and fragments thereof

[0120] The following embodiments apply in the context of the compositions, kits and methods provided herein.

[0121] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3.

[0122] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3.

[0123] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3.

[0124] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3.

[0125] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical with the amino acid sequence depicted in SEQ ID NO: 10, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3 are not changed. In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 10.

[0126] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6.

[0127] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6.

[0128] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0129] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0130] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed. In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0131] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 14.

[0132] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 15.

[0133] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a light chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 16.

[0134] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 17 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0135] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 17.

[0136] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 18 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0137] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 18.

[0138] In one embodiment, the anti-TRBC1 antibody is JOVI.1 .

[0139] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof specifically binds to a region of SEQ ID NO: 21 , preferably to a region comprising N4 and K5 of SEQ ID NO: 21 . In one embodiment, the binding region (epitope) is at least 3 amino acids in length. In one embodiment, the binding region comprises N4 and K5 of SEQ ID NO: 21 and other, non -consecutive amino acids of SEQ ID NO: 21. Hence, in one embodiment the binding region is a non-linear epitope.

[0140] In another embodiment, the binding region comprises N4 and K5 of SEQ ID NO: 21 and is at least 3, at least 4, or at least 5 consecutive amino acids of SEQ ID NO. 21 in length. Hence, in one embodiment, the binding region is a linear epitope.

[0141] In another embodiment, the binding region comprises between 3 and 10 amino acids of SEQ ID NO: 21 including N4 and K5. In some embodiments, the binding region comprises between 5 and 10 amino acids of SEQ ID NO: 21 including N4 and K5. In some embodiments, the binding region comprises between 5 and 8 amino acids of SEQ ID NO: 21 including N4 and K5.

[0142] In one embodiment, anti-TRBC1 antibody or epitope-binding fragment thereof preferentially binds to TRBC1 . Hence, in one embodiment, anti-TRBC1 antibody or epitope-binding fragment thereof has a higher affinity for TRBC1 than for TRBC2. In one embodiment, the dissociation constant (KD) of anti- TRBC1 or epitope-binding fragment thereof when binding to TRBC1 is at least a magnitude of 10 smaller than the KD when binding to TRBC2.

[0143] Anti-TRBC2 antibody and fragments thereof

[0144] The following embodiments apply in the context of the compositions, kits and methods provided herein.

[0145] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3.

[0146] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3.

[0147] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3.

[0148] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3.

[0149] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 12, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3 are not changed. In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical with the amino acid sequence depicted in SEQ ID NO: 12, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3 are not changed.

[0150] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 12.

[0151] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 8, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6.

[0152] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6.

[0153] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0154] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0155] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0156] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0157] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 14.

[0158] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 15.

[0159] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a light chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 16.

[0160] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 19, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed. In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 19 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0161] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 19.

[0162] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 20, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed. In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 20 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0163] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 20.

[0164] In some embodiments, the anti-TRBC2 antibody is JOVI.1 comprising the mutations T28K, Y32F, and A96N in the heavy chain variable region, according to KRABAT numbering.

[0165] In some embodiments, the anti-TRBC2 antibody is JOVI.1 comprising the mutations T28K, Y32F, A96N and N99M in the heavy chain variable region, according to KRABAT numbering.

[0166] In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof specifically binds to a region of SEQ ID NO: 22, preferably to a region comprising K4 and N5 of SEQ ID NO: 22. In one embodiment, the binding region (epitope) is at least 3 amino acids in length. In one embodiment, the binding region comprises K4 and N5 of SEQ ID NO: 21 and other, non -consecutive amino acids of SEQ ID NO: 22. Hence, in one embodiment the binding region is a non-linear epitope.

[0167] In another embodiment, the binding region comprises K4 and N5 of SEQ ID NO: 22 and is at least 3, at least 4, or at least 5 consecutive amino acids of SEQ ID NO. 22 in length. Hence, in one embodiment, the binding region is a linear epitope.

[0168] In another embodiment, the binding region comprises between 3 and 10 amino acids of SEQ ID NO: 22 including K4 and N5. In some embodiments, the binding region comprises between 5 and 10 amino acids of SEQ ID NO: 22 including K4 and N5. In some embodiments, the binding region comprises between 5 and 8 amino acids of SEQ ID NO: 22 including K4 and N5.

[0169] In one embodiment, anti-TRBC2 antibody or epitope-binding fragment thereof preferentially binds to TRBC2. Hence, in one embodiment, anti-TRBC2 antibody or epitope-binding fragment thereof has a higher affinity for TRBC2 than for TRBC1 . In one embodiment, the dissociation constant (KD) of anti- TRBC2 or epitope-binding fragment thereof when binding to TRBC2 is at least a magnitude of 10 smaller than the KD when binding to TRBC1 .

[0170] Antibody combinations

[0171] The following embodiments apply in the context of the compositions, kits and methods provided herein. In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 8.

[0172] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7 and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8.

[0173] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 8.

[0174] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8.

[0175] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical with the amino acid sequence depicted in SEQ ID NO: 10, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3 are not changed; and the anti- TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical with the amino acid sequence depicted in SEQ ID NO: 12, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8 are not changed.

[0176] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 10; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 12.

[0177] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a CDR1 comprising the amino acid sequence depicted in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 comprising the amino acid sequence depicted in SEQ ID NO: 8, a CDR4 comprising the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 comprising the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 comprising the amino acid sequence depicted in SEQ ID NO: 6.

[0178] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , a CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, a CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, a CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, a CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and a CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6.

[0179] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 70% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0180] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 80% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0181] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 90% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0182] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and , the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that is at least 95% identical with the amino acid sequence depicted in SEQ ID NO: 14, wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0183] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 10 and a light chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 14; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 12 and a light chain variable region that comprises the amino acid sequence depicted in SEQ ID NO: 14. In one embodiment, the anti-TRBC1 and the anti-TRBC2 antibody or epitope-binding fragment thereof each comprise a heavy chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 15.

[0184] In one embodiment, the anti-TRBC1 and the anti-TRBC2 antibody or epitope-binding fragment thereof each comprises a light chain constant region that comprises the amino acid sequence depicted in SEQ ID NO: 16.

[0185] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 17 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and the anti-TRBC2 antibody or epitopebinding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 19 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0186] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 18 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 1 , CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 3, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed; and In one embodiment, the anti-TRBC2 antibody or epitope-binding fragment thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical with the amino acid sequence depicted in SEQ ID NO: 20 wherein the sequences CDR1 consisting of the amino acid sequence depicted in SEQ ID NO: 7, CDR2 consisting of the amino acid sequence depicted in SEQ ID NO: 2, CDR3 consisting of the amino acid sequence depicted in SEQ ID NO: 8, CDR4 consisting of the amino acid sequence depicted in SEQ ID NO: 4, CDR5 consisting of the amino acid sequence depicted in SEQ ID NO: 5, and CDR6 consisting of the amino acid sequence depicted in SEQ ID NO: 6 are not changed.

[0187] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 17 or SEQ ID NO: 18; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 19 or 20. In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 17; and the anti-TRBC2 antibody or epitope-binding fragment thereof comprises the amino acid sequence depicted in SEQ ID NO: 19.

[0188] In one embodiment, the anti-TRBC1 antibody is JOVI.1 ; and the anti-TRBC2 antibody is JOVI.1 comprising the mutations T28K, Y32F, and A96N in the heavy chain variable region, according to KRABAT numbering.

[0189] In one embodiment, the anti-TRBC1 antibody specifically binds to a region of SEQ ID NO: 21 , preferably to a region comprising N4 and K5 of SEQ ID NO: 21. In one embodiment, the binding region (epitope) is at least 3 amino acids in length. In one embodiment, the binding region comprises N4 and K5 of SEQ ID NO: 21 and other, non-consecutive amino acids of SEQ ID NO: 21 . Hence, in one embodiment the binding region is a non-linear epitope; the anti-TRBC2 antibody specifically binds to a region of SEQ ID NO: 22, preferably to a region comprising K4 and N5 of SEQ ID NO: 22. In one embodiment, the binding region (epitope) is at least 3 amino acids in length. In one embodiment, the binding region comprises K4 and N5 of SEQ ID NO: 21 and other, non-consecutive amino acids of SEQ ID NO: 22. Hence, in one embodiment the binding region is a non-linear epitope.

[0190] In one embodiment, the anti-TRBC1 antibody or epitope-binding fragment thereof specifically binds to a region of SEQ ID NO: 21 including N4 and K5 of SEQ ID NO: 21 and the anti-TRBC2 antibody or epitope-binding fragment thereof specifically binds to a region of SEQ ID NO: 22 including K4 and N5 of SEQ ID NO: 22.

[0191] Sequences

[0192] Further Embodiments

[0193] 1 . A composition comprising a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI), wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and a second antibody or an epitope-binding fragment thereof specific for a second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

[0194] 2. The composition according to item 1 , wherein the composition reduces cross-reactivity of the first antibody or an epitope-binding fragment thereof compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0195] 3. The composition according to item 1 , wherein the composition enhances specific binding of the first antibody or an epitope-binding fragment thereof to the first variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0196] 4. The composition according to item 1 , wherein the composition reduces non-specific binding of the first antibody or an epitope-binding fragment thereof to the second variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0197] 5. The composition according to any one of the preceding items, wherein the detectable moiety is a chromogenic species or fluorescent species.

[0198] 6. The composition according to any one of the preceding items, wherein the first antibody or an epitope-binding fragment thereof and the second antibody or an epitope-binding fragment thereof are present at a 1 :1 molar ratio.

[0199] 7. The composition according to any one of the preceding items, wherein the composition comprises an excipient or a stabilizer.

[0200] 8. The composition according to any one of the preceding items, wherein the first antibody or epitope-binding fragment thereof is an anti-TRBC1 antibody and the second antibody or epitopebinding fragment thereof is an anti-TRBC2 antibody.

[0201] 9. The composition according to any one of the preceding items, wherein the first antibody or epitope-binding fragment thereof is an anti-TRBC2 antibody and the second antibody or epitopebinding fragment thereof is an anti-TBRC1 antibody.

[0202] 10. Use of the composition according to any one of items 1 -9 in the detection of the first variant of the POI.

[0203] 11 . Use according to item 10, wherein the POI is TRBC.

[0204] 12. Use of the composition according to claim 8, wherein POI is TBRC and the first variant of the POI is TRBCI .

[0205] 13. Use of the composition according to claim 9, wherein POI is TRBC and the first variant of the POI is TRBC2.

[0206] 14. A Composition comprising an anti-TRBC1 antibody or a epitope-binding fragment thereof and an anti-TRBC2 antibody or a epitope-binding fragment thereof, wherein only one of the antibodies is conjugated with a detectable moiety. 15. Use of the composition according to item 1 in the detection of TRBC1 or TRBC2.

[0207] 16. The composition of item 14, wherein the TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0208] 17. The composition of item 14, wherein the TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

[0209] 18. A kit comprising a first composition comprising an anti-TRBC1 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti-TRBC2 antibody or a epitopebinding fragment thereof not conjugated with a detectable moiety; and further comprising a second composition comprising an anti-TRBC2 antibody or a epitope-binding fragment thereof conjugated with a detectable moiety and an anti-TRBC1 antibody or a epitopebinding fragment thereof not conjugated with a detectable moiety.

[0210] 19. The kit of item 18, wherein the first composition and the second composition are comprised in separate vials.

[0211] 20. The kit of item 18, wherein each antibody is comprised in a separate vial.

[0212] 21. In vitro method of enhancing specificity of a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI) in a cell population expressing first variant and a further second variant of the POI in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising a first antibody or an epitope-binding fragment thereof specific for the first variant of the POI, wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety, and a second antibody or an epitope-binding fragment thereof specific for second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each antibody to their respective target epitope; c) detecting the conjugated first antibody or an epitope-binding fragment thereof.

[0213] 22. The in vitro method according to item 21 , wherein the protein of interest is a surface receptor.

[0214] 23. The in vitro method according to any one of items 21 or 22, wherein the first and second variants of the POI are homologs, paralogs, orthologs, isoforms or mutants, 24. The in vitro method according to any one of the preceding items, wherein the first antibody or an epitope-binding fragment thereof specifically binds to a first epitope in the first variant of the POI and the second antibody or an epitope-binding fragment thereof specifically binds to a second epitope in the second variant of the POI, wherein the amino acid sequence of the first epitope and the amino acid sequence of the second epitope are at least 50% identical.

[0215] 25. The in vitro method according to any one of the preceding items, wherein the first epitope and the second epitope are in the same location within the amino acid sequence of the first and second variant of the POI.

[0216] 26. The in vitro method according to any one of the preceding items, wherein the amino acid sequence of first variant of the POI and the amino acid sequence of second variant of the POI are at least 90% identical.

[0217] 27. The in vitro method according to any one of the preceding items, wherein the composition comprising the first antibody or an epitope-binding fragment thereof and the second antibody or an epitope-binding fragment thereof reduces cross reactivity between the first antibody or an epitopebinding fragment thereof and the second variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

[0218] 28. The in vitro method according to any one of the preceding items, wherein the binding of the second antibody or an epitope-binding fragment thereof prevents binding of the first antibody or an epitope-binding fragment thereof to epitopes recognised by the first antibody or an epitope-binding fragment thereof comprised in the second variant.

[0219] 29. In vitro method of enhancing specificity of an anti-TRBC1 antibody in a cell population expressing TRBC1 and TRBC2 in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 conjugated with a detectable moiety and an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2 not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each TRBC antibody to their respective target epitope; c) detecting the detectable moiety.

[0220] 30. In vitro method of enhancing specificity of an anti-TRBC2 antibody in a cell population expressing TRBC1 and TRBC2 in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2 conjugated with a detectable moiety and an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each TRBC antibody to their respective target epitope; c) detecting the detectable moiety. 31 . The in vitro method according to any one of items 29 or 30, wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof and the anti-TRBC2 antibody or an epitope-binding fragment thereof bind to the an epitope sequence that in the same location within the amino acid sequence of TRBC1 and TRBC2.

[0221] 32. The in vitro method according to any one of items 29 to 31 , wherein the amino acid sequence of the antibody-binding epitopes in TRBC1 and TRBC2 have at least 50% sequence identity.

[0222] 33. The in vitro method according to any one of items 29 to 32, wherein the amino acid sequence of the epitope in TRBC1 comprises the amino acids N4 and K5 of the TRBC1 amino acid sequence depicted in SEQ ID NO: 1 and the epitope in TRBC2 comprises the amino acids K4 and N5 of the TRBC2 amino acid sequence depicted in SEQ ID NO: 2.

[0223] 34. The in vitro method according to any one of items 29 to 33, wherein the composition comprising both anti-TRBC1 and anti-TRBC2 antibodies or epitope-binding fragments thereof reduces cross reactivity between the two antibodies or epitope-binding fragments thereof.

[0224] 35. The in vitro method according to any one of items 29 to 34, wherein the detectable moiety is selected from the group consisting of PE, APC, FLAG, HA, His, Myc, V5, fluorescent proteins, PE APC tandem dye, organic or polymer dyes.

[0225] 36. The in vitro method according to any one of items 29 to 35, wherein the detectable moiety is detected using a method selected from the group consisting of flow cytometry, fluorescence- activated cell sorting (FACS), Single-cell analysis, Western blot and Immunostaining, preferably wherein the method is flow cytometry.

[0226] 37. The in vitro method according to any one of items 29 to 36, wherein each antibody or an epitopebinding fragment thereof is selected from the group consisting of a monoclonal antibody, Fab fragment, F(ab') fragment, F(ab')2 fragment, F( ab )c fragment, single chain variable fragment (scFv) and single domain antibody fragment (sdAB).

[0227] Examples

[0228] Antibodies

[0229] Anti-TRBC1 clone JOVI.1 is used in all experiments. Conjugated anti-TRBC1 is conjugated with the flourochrome phycoerythrin (PE). The antibody is derived from mouse and has isotype lgG2a.

[0230] Anti- TRBC2 clone SAM .2 is used in all experiments. Conjugated anti-TRBC2 is conjugated with the fluorochrome allophycocyanin (APC). The antibody is derived from mouse and has isotype lgG1.

[0231] Antibodies used for preparing subpopulations of T-cells are anti-TCR PAN o / p-PE, IP26A (Product No: B49177, Beckman Coulter); CD3-PC7, UCHT1 (Product No:737657, Beckman Coulter), CD4- FITC, 13B8.2 (Product No: A07750, Beckman Coulter), or CD8-APC-A700, B9.11 (Product No: B49181 , Beckman Coulter) ( CD4 and CD8 data not shown).

[0232] Flow Cytometry For each experiment, 1 OOpI of normal whole blood were stained with each antibody conjugate or purified antibody for 15 minutes, lysed (VersaLyse), washed (PBS 1X) and resuspended (PBS1X, 0,1% formaldehyde) before acquisition. In cases of co-incubation of antibodies, a molar ratio of 1 :1 was used. Acquisitions were done on a Navios flow cytometer and analysis performed with Kaluza Software. All reagents, instrument and software are from Beckman Coulter.

[0233] Example 1 : Non-specific reactivity exists between anti-TRBC1 and anti-TRBC2

[0234] Dual dot plot TRBC1 / TRBC2 gated on lymphocytes or T cell sub-population allow discrimination of each TCR cp positive population, however, the shape of both population indicates that crossreactivity exists between TRBC1 / TRBC2 (see Figure 3, exemplarily showing the CD3-positive T cell population).

[0235] Example 2: Addition of unconjugated, purified antibody of the opposite clone reduced crossreactivity

[0236] The addition of unconjugated, purified antibody of the opposite clone into the anti-TRBC conjugate significantly reduced the cross-reactivity without impacting the staining of the positive population (see Figures 4 and 5).

[0237] For example, an anti-TRBC1 conjugate is formulated with anti-TRBC2 purified antibody and vice versa. This formulation enhances the specific staining by reducing the cross reactivity which allows a better identification of the TRBC1 or TRBC2 positive population (see Figures 4 and 5).

[0238] The experiment was also performed on cell lines to further explore cross reactivity: 10OpI of each fresh Cell line diluted at 5.106C / ml in PBS1X, BSA 2mg / ml NaN3 0,01 % were stained 15 minutes at room temperature with :

[0239] - 1 pg of anti-TRBC1-PE + / - anti-TRBC2-PUR or 1 pg of isotypic control lgG2a-PE

[0240] - 0,75pg of anti-TRBC2-APC + / - anti-TRBC1-PUR or 0,75pg of isotypic control lgG1-APC

[0241] PUR refers to a purified, unconjugated antibody. Then samples were washed with 3ml of PBS 1X and centrifuge 5 minutes at 300G. After aspiration of supernatant, the pellet was resuspended in PBS 1X, 0,5% formaldehyde prior acquisition on Navios Ex.

[0242] Figures 6-9 show results of experiments on DAUDI, JURKAT and HPB-ALL cell lines. DAUDI is a B- cell line negative for TRBCI and TRBC2 and serves as a negative control. JURKAT is a T-cell line with clonal expression of TRBC1 . HPB-ALL is a T-cell line with clonal expression of TRBC2. Thus, the reactivity of each antibody can be tested when none or only one antigen are present.

[0243] X-axis indicates fluorescence intensity, y axis indicates count. White histograms indicate background / negative isotypic control staining (Gate A). Black histograms indicate antibody staining (PE or APC labelled, Gate B).

[0244] Figure 6C shows a shift in the histogram of HPB-ALL compared to Figure 6B, showing a loss of fluorescence signal that indicates a reduction in non-specific binding of anti-TRBC1-PE to TRBC2 in HPB-ALL cells (indicated by arrow). The positive signal in JURKAT cells is not affected, as can be seen by the similar relative mean fluorescence intensity (RMFI). Hence, non-specific binding of anti- TRBC1-PE in HPB-ALL cells is reduced by addition of purified, unconjugated anti-TRBC2 antibody.

[0245] Conversely, Figure 8C shows a loss of unspecific signal in JURKAT cells (indicated by arrow) compared to Figure 8B. The positive signal in HPB-ALL cells is not affected. Hence, non-specific binding of anti-TRBC2-APC in JURKAT cells is reduced by addition of purified, unconjugated anti- TRBC1 antibody.

[0246] Figure 7 shows overlay histograms of the isotypic control and anti-TRBC1-PE staining. In the top row, anti-TRBC1-PE was added alone. In the bottom row, anti-TRBC1-PE was added together with purified, unconjugated anti-TRBC2. In the cell line HPB-ALL, which only expresses TRBC2, unspecific signal of anti-TRBC1-PE can be seen in the top row (black histogram). In the bottom row, this unspecific staining is almost completely eliminated. Positive staining in TRBC1 -expressing JURKAT cells is not affected.

[0247] Figure 9 shows overlay histograms of the isotypic control and anti-TRBC2-APC staining. In the top row, anti-TRBC2-APC was added alone. In the bottom row, anti-TRBC2-APC was added together with purified, unconjugated anti-TRBC1. In the cell line JURKAT, which only expresses TRBC1 , unspecific signal of anti-TRBC2-APC can be seen in the top row (black histogram). In the bottom row, this unspecific staining is completely eliminated. Positive staining in TRBC2-expressing HPB- ALL cells is not affected.

Claims

Claims1 . A composition comprising a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI), wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety; and a second antibody or an epitope-binding fragment thereof specific for a second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety.

2. The composition according to claim 1 , wherein the composition reduces cross-reactivity of the first antibody or an epitope-binding fragment thereof compared to a composition comprising only the first antibody or an epitope-binding fragment thereof.

3. The composition according to any one of claims 1 or 2, wherein(i) the detectable moiety is a chromogenic species or fluorescent species;(ii) the first antibody or an epitope-binding fragment thereof and the second antibody or an epitopebinding fragment thereof are present at a molar ratio between 1 :0.1 to 1 :10; and / or(iii) each antibody or an epitope-binding fragment thereof is selected from the group consisting of monoclonal antibody, Fab fragment, F(ab') fragment, F(ab')2 fragment, F( ab )c fragment, single chain variable fragment (scFv) and single domain antibody fragment (sdAB).

5. The composition according to any one of the preceding claims, wherein(i) the first antibody or an epitope-binding fragment thereof is anti-TRBC1 and wherein the second antibody or an epitope-binding fragment thereof is anti-TRBC2; or(ii) the first antibody or an epitope-binding fragment thereof is anti-TRBC2 and wherein the second antibody or an epitope-binding fragment thereof is anti-TRBC1 .

6. The composition according to claim 5, wherein the TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6; and / or wherein the TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, and a CDR3 sequence according to SEQ ID NO: 8, preferably wherein the TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

7. The composition according to any one of claims 1 to 6, wherein the anti-TRBC1 antibody or an epitope-binding fragment thereof and the anti-TRBC2 antibody or an epitope-binding fragmentthereof bind to the an epitope sequence that in the same location within the amino acid sequence of TRBC1 and TRBC2, preferably wherein the amino acid sequence of the epitope in TRBC1 comprises the amino acids N4 and K5 of the TRBC1 amino acid sequence depicted in SEQ ID NO: 1 and the epitope in TRBC2 comprises the amino acids K4 and N5 of the TRBC2 amino acid sequence depicted in SEQ ID NO: 2.

8. Use of the composition according to any one of claims 5 to 7 (i) in the detection of TRBC1 variant of TRBC.

9. Use of the composition according to any one of claims 5 to 7 (ii) in the detection of TRBC2 variant of TRBC.

10. Kit comprising the composition according to any one of claims 5 to 7 (i) and / or the composition according to any one of claims 5 to 7 (ii).

11. In vitro method of enhancing specificity of a first antibody or an epitope-binding fragment thereof specific for a first variant of a protein of interest (POI) in a cell population expressing first variant and a further second variant of the POI in a mutually exclusive pattern, comprising the steps of: a) providing a composition comprising a first antibody or an epitope-binding fragment thereof specific for the first variant of the POI, wherein the first antibody or an epitope-binding fragment thereof is conjugated with a detectable moiety, and a second antibody or an epitope-binding fragment thereof specific for second variant of the POI, wherein the second antibody or an epitope-binding fragment thereof is not conjugated with a detectable moiety; b) contacting the composition with the cell population under conditions that allow binding of each antibody to their respective target epitope; c) detecting the conjugated first antibody or an epitope-binding fragment thereof; preferably wherein the amino acid sequence of first variant of the POI and the amino acid sequence of second variant of the POI are at least 90% identical.

12. The in vitro method according to claim 11 , wherein the composition comprising the first antibody or an epitope-binding fragment thereof and the second antibody or an epitope-binding fragment thereof reduces cross reactivity between the first antibody or an epitope-binding fragment thereof and the second variant of the POI compared to a composition comprising only the first antibody or an epitope-binding fragment thereof; and / or wherein the binding of the second antibody or an epitope-binding fragment thereof prevents binding of the first antibody or an epitope-binding fragment thereof to epitopes recognised by the first antibody or an epitope-binding fragment thereof comprised in the second variant.

13. In vitro method of enhancing specificity of an TRBC-variant specific antibody in a cell population expressing TRBC1 and TRBC2 in a mutually exclusive pattern, comprising the steps of:a) providing a composition according to any one of claims 5 to 7; b) contacting the composition with the cell population under conditions that allow binding of each anti-TRBC antibody to their respective target epitope; c) detecting the detectable moiety.

14. A kit comprising(i) a first composition comprising an anti-TRBC1 antibody or an epitope-binding fragment thereof specific for TRBC1 conjugated with a detectable moiety and an anti-TRBC2 antibody or an epitopebinding fragment thereof specific for TRBC2 not conjugated with a detectable moiety; and(ii) a second composition comprising an anti-TRBC2 antibody or an epitope-binding fragment thereof specific for TRBC2 conjugated with a detectable moiety and an anti-TRBC1 antibody or an epitopebinding fragment thereof specific for TRBC1 not conjugated with a detectable moiety.

15. The kit according to claim 14, wherein the TRBC1 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 1 , a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 3, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6; and wherein the TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, and a CDR3 sequence according to SEQ ID NO: 8, preferably wherein the TRBC2 antibody or an epitope-binding fragment thereof comprises a CDR1 sequence according to SEQ ID NO: 7, a CDR2 sequence according to SEQ ID NO: 2, a CDR3 sequence according to SEQ ID NO: 8, a CDR4 sequence according to SEQ ID NO: 4, a CDR5 sequence according to SEQ ID NO: 5, and a CDR6 sequence according to SEQ ID NO: 6.

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