Antibodies that specifically bind mucosal associated invariant t (MAIT) cells

Abstract

The present invention relates to the field of immunology, and more specifically to antibodies that are useful in the medical field with improved properties, their composition, production methods and uses. In particular, the present invention provides antibodies that specifically bind to the Vα7.2 chain of the TCR on Mucosal Associated Invariant T (MAIT) cells. It also provides multispecific antibody constructs, methods for producing them, and uses thereof.

Description

[0001] Antibodies that specifically bind Mucosal Associated Invariant T (MAIT) cells

[0002] The present invention relates to the field of immunology, and more specifically to antibodies that are useful in the medical field with improved properties, their composition, production methods and uses.

[0003] BACKGROUND OF THE INVENTION

[0004] Recent advancements for the development of cancer immunotherapies have been made through T cell redirection approaches using bispecific antibodies (BsAbs). These antibodies simultaneously bind T cells and tumor cells, recruiting the former to the latter (van de Donk and Zweegman, 2023). This leads to the formation of an immunological synapse, activation of the T cell and release of cytotoxins, ultimately resulting in lysis of the target cell.

[0005] Mucosal Associated Invariant T (MAIT) cells are an unconventional subtype of T-cells present in blood, where they represent up to 20% of the cc|3 T-cells. They are found abundantly in almost all organs, and are particularly enriched in mucosal tissues such as lung, liver, skin and colon where they play an important function in barrier immunity and in the response to bacterial and viral infections. MAIT cells can be activated via both a TCR-dependent and TCR-independent manner. Upon activation, MAIT cells rapidly produce multiple cytokines such as interferon-y (INF-y), tumor necrosis factor a (TNF-a) and cytolytic products such as granzyme B and perforin. The MAIT TCR recognizes bacterial metabolites presented in the context of MR1 (MHC -related protein 1) expressed on the surface of an infected cell and can directly kill the cell via formation of an immunological synapse. MAITs have also been shown to kill cancer cells that present MRI on their surface (Dusseaux et al., 2011).

[0006] While T lymphocytes typically display a wide diversity of T cell receptors (TCR), MAIT cells display a semi-invariant cc|3 TCR with very limited diversity. More precisely, all MAIT cells express the alpha chain Va7.2 that is mostly associated with the segment Joc33 (about 77%) whilst some cells incorporate the Joc2O or Jex 12 segment and this unique alpha chain is associated with a restricted repertoire of beta chains to create the functional MAIT TCR. (Tilloy et al., 1999; Reantragoon et al., 2013). In addition to the Va7.2 TCR element, MAIT cells highly express the CD 161 marker and the co-expression of these two proteins on a T-cell (Va7.2+, CD161hi) can be used to identify the vast majority of MAIT cells. An antibody (or antigen binding agent), named 3C10, that specifically binds an epitope present in Va7.2-Ja33 has been described in international patent application W02008 / 087919.

[0007] MAIT cells can be redirected to kill tumor cells through a BsAb comprising an anti-Va7.2 domain and a domain that specifically binds to a tumor associated antigen (TAA), as described in international patent application W02021 / 140190. An anti-Va7.2 domain will bind to all MAIT cells and BsAbs comprising such a domain and a TAA binding domain are termed MAIT engagers.

[0008] Ideally, the part of a BsAb that binds to T-cells must have both affinity for the TCR of interest and specificity to that TCR. Typically, specificity encompasses binding with far greater affinity to the TCR of interest than to any other TCR or any other substance. Thus, a BsAb that binds Va7.2 with high specificity and affinity will recruit all MAIT cells and redirect them to kill cancer cells expressing a particular TAA. Restricting binding to the Va7.2 segment of the TCR prevents the widespread activation of the majority of CD4 and CD8 cc|3 T cells. Recruitment and activation of CD4+ T cells, for example, may elicit a cytokine storm, and activation of regulatory T cells (Tregs) may increase immune suppression in the tumor microenvironment and support tumor growth (Koristka et al, 2012). Thus, specific recruitment of MAIT cells to tumors via a BsAb MAIT engager, incorporating a Va7.2 segment binding domain is expected to reduce side effects and increase patient tolerance to treatment.

[0009] It should also bind in vivo to the desired T cells (i.e., cells which display the TCR of interest), and these T cells should be activated when presented with the domain of the antibody that binds to the TCR. Furthermore, cross -reactivity with the desired TCR in non-human mammalian model species may be desirable, in particular non-human primate (NHP) as it is a model used for preclinical toxicity studies. Greene et al. showed that 3C10, the only anti-Va7.2 antibody available to date, has variable cross-reactivity to NHPs and doesn’t bind by flow cytometry to M. mulatto (Rhesus macaque) or M. fascicularis (cynomolgus monkey) that are the main NHP models used in preclinical toxicity studies.

[0010] A major concern with antibodies of non-human origin is that they are often immunogenic, thereby limiting their effectiveness and, in some cases, causing dangerous allergic reactions. The immune response to such foreign antibodies includes the production of specific, high affinity antibodies which bind to and effect elimination of the foreign antibodies, thereby substantially reducing the antibodies’ effectiveness by promoting their clearance from the body and inhibiting its ability to bind to the targeted antigen. To overcome this problem, it is possible to humanize non-human antibodies of interest to reduce immunogenicity to humans, while retaining the specificity and affinity to the desired antigen. Such humanized antibody typically comprises one or more variable domains in which the antigen binding domains are derived from the non-human antibody, and framework regions derived from human or humanized antibody sequences.

[0011] The present invention relates to novel monoclonal anti-Voc7.2 antibodies showing high affinity and specificity to human Voc7.2-containing TCRs and human MAIT cells and showing significant cross-reactivity to NHP Voc7.2-containing TCR.

[0012] SUMMARY OF THE INVENTION

[0013] The inventors have identified two monoclonal antibodies that specifically bind to the Va7.2 chain of the TCR on human MAIT cells. Those antibodies, named Mabl7 and Mabl8, have been tested for their affinity and specificity with respect to the Va7.2 chain of the TCR MAIT cells, and have shown improved properties compared to the 3C10 antibody, as set forth in the Experimental section.

[0014] On that basis, the invention provides an antigen-binding fragment that specifically binds to a polypeptide comprising Va7.2, wherein said antigen binding fragment comprises the following heavy chain complementarity-determining regions (H-CDRs): i)

[0015] H-CDR1 that shows sequence SEQ ID NO: 1 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 1, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1;

[0016] H-CDR2 that shows sequence SEQ ID NO: 2 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 2, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 2; and

[0017] H-CDR3, that shows SEQ ID NO: 3 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 3; or ii)

[0018] H-CDR1 that shows sequence SEQ ID NO: 4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 4, and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NO: 4; H-CDR2 that shows sequence SEQ ID NO: 5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 5; and

[0019] H-CDR3 that shows sequence SEQ ID NO: 6 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 6, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

[0020] In a particular embodiment, the antigen binding fragment further comprises : in i), framework regions that show at least 80% identity, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with the framework regions set forth in SEQ ID NO: 7 to 9 and SEQ ID NO: 12; or, in ii), framework regions that show at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with the framework regions set forth in SEQ ID NO: 8 and SEQ ID NO: 10 to 12, respectively.

[0021] In a particular embodiment, the antigen binding fragment comprises : in (i), a heavy chain variable region sequence as set forth in SEQ ID NO: 13, or having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 ; or in (ii), a heavy chain variable region sequence as set forth in SEQ ID NO: 14, or having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 14.

[0022] In a particular embodiment, the antigen binding fragment further comprises a light chain comprising the following complementarity-determining regions (L-CDRs) : in i),

[0023] - L-CDR1 that shows sequence SEQ ID NO: 15 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 15, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 15; - L-CDR2 that shows sequence SEQ ID NO: 16 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 16, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 16; and

[0024] - L-CDR3, that shows SEQ ID NO: 17 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 17 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 17; or, in ii)

[0025] - L-CDR1 that shows sequence SEQ ID NO: 18 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 18, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 18;

[0026] - L-CDR2 that shows sequence SEQ ID NO: 19 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 19, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19; and

[0027] - L-CDR3, that shows SEQ ID NO: 20 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 20 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 20, respectively ; optionally wherein the light chain further comprises : in i), framework regions that show at least 80% identity, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with the framework regions set forth in SEQ ID NO: 21 to 24; or, in ii), framework regions that show at least 80% identity, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with the framework regions set forth in SEQ ID NO: 25, 26, 23 and 28 respectively.

[0028] In a particular embodiment, the antigen binding fragment comprises : in i), a light chain variable region sequence as set forth in SEQ ID NO: 29, or having at least 80%, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 29 ; or, in ii), a light chain variable region sequence as set forth in SEQ ID NO: 30, or having at least 80%, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 30. In a particular embodiment, the antigen binding fragment is humanized. Preferably, said humanized antigen binding fragment comprises :

[0029] - in i) H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, SEQ ID NO: 78, SEQ ID

[0030] NO: 79 or SEQ ID NO: 80, and H-CDR3 of SEQ ID NO: 3, or variants thereof having at least 80% identity to SEQ ID NOs: 1-3 and 78-80 ; and / or L-CDR1 of SEQ ID NO: 15 or SEQ ID NO: 85, L-CDR2 of SEQ ID NO: 16 or SEQ ID NO: 86, and L-CDR3 of SEQ ID NO: 17, or variants thereof having at least 80% identity to SEQ ID NOs: 15-17 and 85-86 ; or

[0031] - in ii), H-CDR1 of SEQ ID NO: 4 or SEQ ID NO: 81, H-CDR2 of SEQ ID NO: 5, SEQ

[0032] ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 91, and H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92, or variants thereof having at least 80% identity to SEQ ID NOs: 4-6; 81-84 and 91-92 ; and / or L-CDR1 of SEQ ID NO: 18, SEQ ID NO: 87, L- CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20 or SEQ ID NO: 93 ; or variants thereof having at least 80% identity to SEQ ID NOs: 18-20, 87 and 93; respectively ; preferably wherein said humanized antigen binding fragment comprises :

[0033] - in i), a variable heavy chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34 and 35, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 31-35 ; and / or a variable light chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 36, 37, 38 and 39, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 36- 39 ; or

[0034] - in ii), a variable heavy chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 40, 41, 42, 43, 44, 45 and 89 or variants thereof having at least 80% sequence identity to SEQ ID NOs: 40-45 and 89; and / or a variable light chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 90, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 46-50 and 90 ; respectively.

[0035] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 4-6 or having 1, 2 or 3 amino acid modifications with respect to SEQ ID NOs: 4-6 ; and / or L-CDR1 of SEQ ID NO: 87, L- CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 87 and 19-20 or having 1, 2 or 3 amino acid modifications with respect to SEQ ID NOs: 87 and 19-20, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 43 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 49, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 49.

[0036] In a preferred embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91 and H-CDR3 of SEQ ID NO: 92, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 4, 91 and 92 or having 1, 2 or 3 amino acid modifications with respect to SEQ ID NOs: 4, 91 and 92 ; and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 93, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 87, 19 and 93 or having 1, 2 or 3 amino acid modifications with respect to SEQ ID NOs:87, 19 and 93, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 89, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 89 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 90, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 90.

[0037] A further aspect of the invention is a protein construct which comprises the antigen binding fragment as described herein.

[0038] In a particular embodiment, the protein construct comprises the antigen binding fragment as defined above, optionally linked to at least another polypeptide; optionally wherein the protein construct is a monoclonal antibody, a multivalent antibody or a multispecific antibody; preferably wherein the protein construct is an antibody that comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl, IgG2, IgG3 or IgG4 heavy chain constant domain (CHI).

[0039] In a particular embodiment, the protein construct comprises :

[0040] - two identical antigen-binding arms each comprising, or consisting of, an antigen-binding fragment as defined above ; and - an Fc region.

[0041] In particular, the construct may be a multivalent antibody or a multispecific antibody.

[0042] A further aspect of the invention is a multispecific molecule, preferably a bispecific molecule, that comprises the antigen binding fragment as defined above, or the protein construct as defined above, wherein the molecule also comprises at least one domain that binds another antigen, preferably a tumor associated antigen (TAA). In a particular embodiment, the TAA is selected from the group consisting of CD 19, EGFR, and HER2.

[0043] According to such approach, the invention provides a T cell redirection that targets immune cells utilizing the Va7.2 chain in the TCR which is invariant in the Mucosal Associated Invariant T (MAIT) cells and redirects these specific T cells to kill tumor cells. More particularly, the invention provides a multispecific molecule capable of simultaneous binding to a MAIT cell and to a tumor cell, which multispecific molecule comprises at least one anti- Va7.2 domain, i.e. a domain that specifically binds a Voc7.2 TCR, and at least one anti-tumor associated antigen domain (TAA), i.e. a domain that specifically binds a TAA.

[0044] According to the invention, the crosslinking of the T cell receptor by means of such multispecific molecule activates the MAIT cells to kill tumor cells.

[0045] This approach has the advantages of: a) activating only a sub-population of cytotoxic cells against the target cells, b) not activating CD4+ T cells, hence less risk for a cytokine storm and autoreactivity, and c) not activating Tregs to increase their immunosuppressive activity in the tumor microenvironment. Furthermore, since MAIT cells are abundant within human peripheral tissues, particularly in the liver and mucosal tissues, such as lung and gut, migration of MAIT cells into solid tumors from the surrounding healthy tissue could occur in response to primary MAIT- induced killing of the tumor.

[0046] Such constructs are thus particularly useful in treating cancers.

[0047] In a particular embodiment, the multispecific molecule comprises at least one multispecific antigen-binding fragment comprising at least two Fab fragments with different CHI and CL domains, wherein said Fab fragments are tandemly arranged in any order, the C-terminal end of the CHI domain of a first Fab fragment being linked to the N-terminal end of the VH domain of the following Fab fragment through a polypeptide linker, wherein at least one Fab fragment comprises the antigen-binding fragment as described above, and at least another Fab fragment binds another antigen, preferably a TAA, more preferably a TAA selected from the group consisting of CD 19, EGFR, and HER2.

[0048] It is further provided a nucleic acid that encodes the antigen binding fragment and protein constructs comprising these antigen binding fragments as described herein, a host cell containing said nucleic acid, and a method for producing the antigen binding fragment and derivative protein constructs, comprising a step of culturing a host cell as described herein and optionally a step of isolating the antibody or antigen-binding fragment.

[0049] A further aspect of the invention is an isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the antibody, or the antigen-binding fragment, or the protein construct or the multispecific molecule as defined herein.

[0050] A further aspect of the invention is a vector comprising said isolated nucleic acid molecule or said group of isolated nucleic acid molecules.

[0051] A further aspect of the invention is a host cell comprising said isolated nucleic acid molecule and / or said group of isolated nucleic acid molecules and / or said vector.

[0052] A further aspect of the invention is a method for producing the antigen-binding fragment, protein construct or multispecific molecule of the invention, comprising a step of culturing a said host cell and optionally a step of isolating the antigen-binding fragment, protein construct or multispecific molecule.

[0053] A further aspect of the invention is the multispecific molecule as defined herein, for use as a medicament, in particular for use in treating a cancer.

[0054] LEGENDS TO THE FIGURES

[0055] Schematic representation of a bispecific antibody (“BiXAb”) of the invention. Figure 2A: Binding of twenty specimens of mAb generated against Va7.2 chain to an irrelevant TCR using the Vocl2 / V|36 recombinant protein. The y-axis represents Absorbance at 450 nm, the x-axis represents the different antibodies tested at 100 nM.

[0056] Figure 2B: Binding of twenty specimens of mAb generated against Va7.2 chain to MAIT TCR using the Voc7.2 / V|36 recombinant protein. The y-axis represents Absorbance at 450 nm, the x- axis represents the different antibodies tested at 100 nm.

[0057] Figure 3A: Binding profile of five different anti-Va7.2 antibodies to the recombinant human Va7.2Vpi3 TCR, in comparison with the 3C10 and isotype. The y-axis represents Absorbance at 450nM, the x-axis represents the range of antibody concentration in nm.

[0058] Figure 3B: Binding profile of five different anti-Va7.2 antibodies to the recombinant human Va7.2V|32 TCR, in comparison with the 3C10 and isotype. The y-axis represents Absorbance at 450nM, the x-axis represents the range of antibody concentration in nM.

[0059] Figure 4: Gating strategy of MAIT cells using minimal identification markers. (Live dead cell yellow, anti-CD8-BV421, anti-CD3-PercP, anti-Va7.2-FITC, anti-CD161-APC and MR1-5- OPRU tetramer- PE).

[0060] Figure 5A: binding histogram of anti-Va7.2 antibodies overlay with the isotype at 100 nM among MAIT cells (CD161H1CD8+ cells) from 4 human healthy donors and their respective phenotyping for Va7.2+ frequency.

[0061] Figure 5B: binding histogram of anti-Va7.2 antibodies overlay with the isotype at 100 nM among T-cells (CD3+ cells) from 4 human healthy donors depleted in Va7.2 cells and their respective phenotyping for Voc7.2+ frequency.

[0062] Figure 6 Binding profile of exemplary two anti-Voc7.2 antibodies to primary MAIT cells isolated from 4 healthy donors. The y-axis represents the mean of fluorescence of PE signal among CD8+CD161H1cells, the x-axis represents the range of antibody concentration in nM.

[0063] Figure 7. Competition Assay of two anti-Va7.2 antibodies with 3C10 clone for their binding to the recombinant Va7.2 TCR by ELISA. Cross-reactivity of two anti-Voc7.2 antibodies to the recombinant Non-Human

[0064] Primate Va7.2 TCR, in comparison with the 3C10.

[0065] Figure 9: Simultaneous binding of the BiXabs towards the human Va7.2 TCR and HER2 recombinant proteins by DUAL ELISA.

[0066] Figure 10: Potency in inducing tumor cell lysis of two BiXAb HER2xMAIT engager from nonstimulated CD8+ T cells towards human SKBR3 cell line. 11 Potency in degranulation of MAIT cells compared to conventional CD8+T cells in presence of OVCAR3 cell line of an exemplary of two BiXAbs HER2x Va7.2 MAIT engager, from non-stimulated CD8+-T cells. Potency in activation of MAIT cells via the upregulation of CD69 markers compared to conventional CD8+T cells in presence of OVCAR3 cell line of an exemplary of two BiXAbs HER2x Va7.2 MAIT engager, from non-stimulated CD8+-T cells. Potency in activation of MAIT cells via the upregulation of CD 137 markers compared to conventional CD8+T cells in presence of OVCAR3 cell line of an exemplary of two BiXAbs HER2x Va7.2 MAIT engager, from non-stimulated CD8+-T cells. Potency secretion of INFyfrom non-stimulated CD8+ cells redirected by exemplary of BiXAbs HER2x Va7.2 MAIT engager in presence of OVCAR3 cell line by ELISA dosage. Potency of inducing specific MAIT cells proliferation and no CD8+ conventional

[0067] T cells in presence of OVCAR3 cell line from BiXAb HER2 x Va7.2 engager at optimal concentration (InM). Potency of inducing MAIT cells proliferation in presence of OVCAR3 cell line from BiXAb HER2 x Va7.2 engager depending on the protein binding concentration.

[0068] 15: Structural representation of the Epitope of mAbl7 and mAbl8 antibodies on MAIT

[0069] TCR antigen determined by Deep Mutational Scanning using Yeast Surface Display.

[0070] Binding profile of mAbl8 variants to recombinant human TCR. Binding profile of mAbl8 variants to MAIT cells.

[0071] 17: Superior degranulation of MAIT cells induced by mAbl8 variants compared to the

[0072] 3C10 antibody.

[0073] 18: Superior capacity of mAbl8 variants to induce MAIT cells activation compared to the 3C10 antibody.

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] Definitions

[0076] For better understanding of the present invention, certain terms are defined thereafter.

[0077] Additional definitions are presented throughout the detailed description.

[0078] Mucosal associated invariant T (MAIT) cells are non-conventional T cells that are not restricted by classical MHC and are found in blood and tissues, where they contribute to barrier immunity. They have the potential to kill bacterial infected cells or tumor cells based upon expression studies (Salon et al, 2019) and in vitro assays (Le Bourhis et al, 2013). MAIT cells express a semi-invariant TCR (named Va7.2) which recognizes Vitamin B2 precursors presented by the highly evolutionarily conserved MHC class lb molecule, MR1 (Franciszkiewicz et al, 2016; Salou et al, 2017).

[0079] The MAIT cells represent up to 20% of T cells in the blood, but also reside in organs and mucosal tissues such as lung, liver, skin and the colon. Moreover, upon activation MAIT cells can secrete cytotoxic granules such as those containing Granzyme B and produce IFNy and TNFa that contribute to their cytotoxic activity (Dusseaux et al, 2011).

[0080] Va7.2 is the unique alpha chain used in the T cell receptors expressed by MAIT cells. The term includes Voc7.2-Ja33, Voc7.2-Jcx2O or Voc7.2-Jcxl2 alpha chains as these J regions are found together with the Vcx7.2 segment in the intact MAIT TCR. In humans, they consist of TRAV1- 2 joined to TRAJ33, TRAJ20 or TRAJ 12 gene segment respectively with little to no nucleotide additions at the TCR-a complementarity determining region 3 (CDR3a) junction. The amino acid sequence of human and NHP TCR are described in Example 5 based on published data from Tilloy et al, 1999, Reantragoon et al, 2013, Green et al 2016. A more recent analysis of the canonical MAIT TCR-a, in line with those previous reports, highlighted that they are largely germline encoded, although some sequence variation was permitted at positions 90-9 la for TRAJ12+ and TRAJ33+ transcripts and 91-93a for TRAJ20+ transcripts.

[0081] This TCR ex chain pair with a restricted range of TCR-|3 chains, predominantly utilizing the TRBV6 and TRB20 gene families corresponding to the V|313 and Vf>2 chain respectively (Reantragoon et al 2013. Tilloy et al 1999).

[0082] An antibody or antigen-binding fragment “specifically binds” to a target antigen if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. “Specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. Preferably the molecule will not show any significant binding to ligands other than its specific target (e.g., an affinity of about 100-fold less), i.e. minimal crossreactivity. An “antigen-binding fragment” as used herein comprises a portion of an antibody molecule, generally the antigen binding site or variable region that specifically binds to the antigen. Examples of antibody fragments include Fab, Fab', Fab'-SH, F (ab') 2, and Fv fragments, and mutants thereof; molecules comprising an antibody portion; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv molecules (2) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety.

[0083] A “Fab” fragment contains the constant domain of the light chain (CE) and the first constant domain (CHI) of the heavy chain, in addition to the variable domains (VE and CE domains).. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than an intact antibody (see, e.g. , Wahl et al, 1983).

[0084] An "Fv" fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VE dimer).

[0085] It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VE dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some instances even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) can have the ability to recognize and bind target, although at a lower affinity than the entire binding site.

[0086] "Single-chain Fv" or "scFv" antibody binding fragments comprise the VH and VE domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VE domains which enables the scFv to form the desired structure for target binding. As used herein, a “single-domain antibody” (sdAb) also known as nanobody (Nb) refers to a single-variable domain, derived from a heavy-chain only antibody, which is able to bind an antigen, an epitope or a ligand alone, that is to say, without the requirement of another binding domain. A single domain antibody may derive from, or consists in, a VHH that refers to a single variable domain found in heavy-chain antibodies of Camelidae.

[0087] As used herein, the term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g. chimeric antibodies that contain minimal sequence derived from a non-human antibody). A “humanized form” of an antibody, e.g., a non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non- human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by framework region residues from the donor antibody. Alternatively, selected framework region residues of the donor antibody are replaced by framework region residues from a human or humanized antibody. Additional framework region modifications may be made within the human framework sequences. Humanized antibodies thus may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such amino acid modifications may be made to further refine antibody function and / or increase the humanization process.

[0088] As used herein, by “amino acid modification” is meant a change in the amino acid sequence of a polypeptide. "Amino acid modifications" which may be also termed "amino acid changes", herein include amino acid mutations such as substitution, insertion, and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. Generally, more substitutions can be made in FR regions, in contrast to CDR regions, as long as they do not adversely impact the binding function of the antigen binding fragment (such as reducing the binding affinity by more than 50% as compared to the original antigen binding fragment). In some embodiments, the modified antigen binding fragment has the same binding specificity and has at least 50% of the affinity of the original antigen binding fragment.

[0089] As used herein, “parent sequence” refers to an unmodified polypeptide that is subsequently modified to generate a variant. “Variant sequence”, “sequence variant” or “variant”, as used herein, refers to a sequence that differs from that of a parent sequence by virtue of at least one amino acid modification. In some embodiments, the variant may comprise one or several conservative substitutions. In some further embodiments, the variant may comprise one or several amino acid modifications in the CDR domains of the parent fragment. In some other embodiments, the variant of the parent construct may comprise one or several amino acid modifications in at least one framework domain. Humanized versions are encompassed in such variants. Preferably, the variants show at least 80%, preferably at least 85%, still preferably at least 90%, 92%, 95%, 96%, 97%, 98% or 99% homology or identity sequence with the native sequence. In a particular embodiment, the variants of the invention are functional variants, i.e. the mutations do not substantially impact the function of the antibody. The functional variants of the invention are capable of binding to Va7.2. In some examples, the variants possess similar antigen binding affinity relative to the reference antibody. The affinity of the binding is defined by the terms ka (associate rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). In a particular embodiment, the variant has substantially similar binding activities (e.g., affinity and / or specificity) and bioactivities (such as cross -reactivity) relative to the reference antibody. In some embodiments, the variant has the same binding specificity and has at least 50%, 60%, 70%, 80%, or at least 90% of the affinity of the original antibody. In a particular embodiment, the functional variants of the antibody of the invention are capable of binding to the same of substantially the same epitope on Va7.2 TCR. In a particular embodiment, the functional variants of the “mAbl7” antibody of the invention are capable of binding to following residues of Va7.2 TCR: L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66. In another particular embodiment, the functional variants of the “mAbl8” antibody of the invention are capable of binding to the following residues of Va7.2 TCR: L51, D52, K68, G53, L54, F63, S62, E55 and R66.

[0090] In a particular embodiment, the antigen-binding fragments described herein may be capable to bind to substantially the same epitope as antibody “mAbl7” or “mAbl8” described in the present application. When an antibody or agent is said to "compete" or "bind to substantially the same epitope" as a particular antibody, it means that the antibody or agent competes with said antibody in a binding assay using either recombinant molecules or surface expressed molecules. For example, if a test antibody or agent reduces the binding of mAbl8 antibody to a Voc7.2-Jcx33 polypeptide in a binding assay, the antibody or agent is said to "compete" with mAbl8 antibody.

[0091] The “identity” or the "percentage identity" between two amino acid sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for example, using the algorithm for global alignment of Needleman- Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the proportion of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using a sequence analysis software. Tools, software and algorithms for determination of the percentage of identity between two amino acid sequences are generally well-known and commonly employed by those skilled in the art.

[0092] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to a mammal being assessed for treatment and / or being treated. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, rabbit, dog, non-human primate etc.

[0093] The term “treatment” or “treating” refers to an action, application or therapy, wherein a subject, including a human being, is subjected to medical aid with the purpose of improving the subject's condition, directly or indirectly. Particularly, the term refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or a combination thereof in some embodiments. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. For example, with respect to cancer, “treatment” or “treating” may refer to slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof.

[0094] “Cross-reactivity”, in a broad sense, refers to the ability of a compound to elicit reactions outside of the main expected reaction. In immunology and in particular where antigen-binding fragments or antibodies are concerned, cross -reactivity is often used to describe an antigenbinding fragment or antibody that is able to bind a target other that the preferred antigen. Crossreactivity is used, in particular, when describing an antigen-binding fragment or antibody that is able to bind both an antigen found in humans, and the equivalent of said antigen in a mammal species, in particular a model species, for example encompassing but not limited to : rhesus monkey, cynomolgus monkey, baboon, chimpanzee, marmoset, pig, canine species, or mouse. Reactivity or binding of an antigen-binding fragment or antibody to an antigen can be measured be methods well known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0095] As used herein, the term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid or some combination thereof) that is expressed (or overexpressed relative to normal tissues) on the surface of a cancerous cell, either entirely or as a fragment (e.g., MHC / peptide). As used herein, the term "cancerous cell" refers to a cell that is undergoing or has undergone uncontrolled proliferation. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, e.g., CD19. In some embodiments, a TAA is a cell surface molecule that is overexpressed in a cancerous cell in comparison to a normal cell, for instance, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell / tissue. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in the cancerous cell, for instance, a molecule that contains deletions, additions or mutations (e.g. EGFRvIII) in comparison to the molecule expressed on a normal cell. In some embodiments, a TAA will be expressed exclusively on the cell surface of a cancerous cell, entirely or as a fragment (e.g., MHC / peptide), and not synthesized or expressed on the surface of a normal cell. Accordingly, the term "TAA" encompasses cell antigens that are specific to cancer cells, sometimes known in the art as tumor- specific antigens ("TSAs"). The anti-Va7.2 binding fragments

[0096] The antigen-binding fragments described herein specifically bind to Voc7.2, in particular to human and NHP Voc7.2, i.e. they comprise at least one domain that binds Va7.2, e.g. Voc7.2- Ja33, Voc7.2-Jcx2O and / or Voc7.2-Jocl2.

[0097] In a particular embodiment, the antigen-binding fragments described herein are capable of competing with monoclonal antibody 3C10 described in international patent application W02008 / 087219.

[0098] Typically, the antigen-binding fragments may display improvements in one or more properties that are desirable for antigen-binding fragments when compared to 3C10, such as in vitro binding affinity to Va7.2 , specificity to Va7.2, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, solubility, cross -reactivity to the Voc7.2 of non-human primate model species or increased capacity to bind and activate Voc7.2-presenting cells, in particular human MAIT cells.

[0099] More particularly, the invention provides an antigen binding fragment that specifically binds to Voc7.2, wherein said antigen binding fragment comprises the complementarity determining regions H-CDR1, H-CDR2 and H-CDR3 as described below, or variants thereof.

[0100] In preferred embodiments, said variants have at least 80%, preferably 85, 90, 95, 97, 98 % sequence identity with the parent sequence (SEQ ID NO: 1, 2 and / or 3, or SEQ ID NO: 4, 5 and / or 6 respectively), and / or have a substitution of one, two or three amino acids with said parent sequence.

[0101] H-CDR1 is (i) GYYLH (SEQ ID NO: 1) or (ii) DYYMN (SEQ ID NO: 4);

[0102] H-CDR2 is (i) RVNPNNGDTRYNQKFKG (SEQ ID NO: 2) or (ii) RINPSNGDTFYNQKFKG (SEQ ID NO: 5), respectively;

[0103] H-CDR3 is (i) GGDGMDY (SEQ ID NO: 3) or (ii) WGDSMDY (SEQ ID NO: 6), respectively.

[0104] In a particular embodiment, the antigen binding fragment comprises the H-CDR1 of SEQ ID NO:1, H-CDR2 of SEQ ID NO:2 and H-CDR3 of SEQ ID NOG, or variants having at least 80%, preferably 85, 90, 95, 97, 98 % sequence identity with SEQ ID NOs: 1, 2 and 3, and / or having a substitution of one, two or three amino acids with SEQ ID NOs: 1, 2 and 3. In another particular embodiment, the antigen binding fragment comprises the H-CDR1 of SEQ ID NO:4, H-CDR2 of SEQ ID NO:5 and H-CDR3 of SEQ ID NO:6, or variants having at least 80%, preferably 85, 90, 95, 97, 98 % sequence identity with SEQ ID NOs: 4, 5 and 6, and / or having a substitution of one, two or three amino acids with SEQ ID NOs: 4, 5 and 6.

[0105] In a particular embodiment, the antigen binding fragment comprises a variable heavy chain region comprising H-CDR1, H-CDR2 and H-CDR3 as described above, or variants thereof, and further comprising framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NO:7 to SEQ ID NO: 12.

[0106] In a particular embodiment, the antigen binding fragment comprises framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions EIQLQQSGPELVKPGASVKISCKASGYSFT (SEQ ID NO: 7), WVKQSHGKSLEWIG (SEQ ID NO: 8), RAILTVDKSSSTAYMELRSLTSEDSAVYYCAR (SEQ ID NO: 9) and WGQGTSVTVSS (SEQ ID NO: 12). Preferably, the antigen binding fragment comprises framework regions consisting of EIQLQQSGPELVKPGASVKISCKASGYSFT (SEQ ID NO: 7), WVKQSHGKSLEWIG (SEQ ID NO: 8),

[0107] RAILTVDKSSSTAYMELRSLTSEDSAVYYCAR (SEQ ID NO: 9) and WGQGTSVTVSS (SEQ ID NO: 12).

[0108] In another particular embodiment, the antigen binding fragment comprises framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions EVQLQQSGPELVKPGASVKISCKASGYSFT (SEQ ID NO: 10), WVKQSHGKSLEWIG (SEQ ID NO: 8), KATLTVDKSSSTAHMELLSLTSEDSAVYYCGR (SEQ ID NO: 11), and WGQGTSVTVSS (SEQ ID NO: 12) respectively. Preferably, the antigen binding fragment comprises framework regions consisting of EVQLQQSGPELVKPGASVKISCKASGYSFT (SEQ ID NO: 10), WVKQSHGKSLEWIG (SEQ ID NO: 8), KATLTVDKSSSTAHMELLSLTSEDSAVYYCGR (SEQ ID NO: 11), and WGQGTSVTVSS (SEQ ID NO: 12)

[0109] In a preferred embodiment, the antigen binding fragment comprises, or consists of, a heavy chain variable region sequence as set forth in :

[0110] EIOLOQSGPELVKPGASVKISCKASGYSFTGYYLHWVKQSHGKSLEWIGRVNPNNG VTVSS (SEQ ID NO: 13) or

[0111] EVOLOQSGPELVKPGASVKISCKASGYSFTDYYMNWVKOSHGKSLEWIGRINPSNG

[0112] VTVSS (SEQ ID NO: 14), wherein underlined and bold sequences represent the three CDR sequences.

[0113] In a particular embodiment, the antigen binding fragment comprises, or consists of, a heavy chain variable region sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14. In a particular embodiment, the amino acid modification(s) with respect to SEQ ID NO: 13 or SEQ ID NO: 14 are not present in the CDR sequences.

[0114] In a particular embodiment, the antigen-binding fragment as described above is a single-domain antibody.

[0115] In another embodiment, the antigen-binding fragment further comprises a variable light chain comprising the complementarity determining regions L-CDR1, L-CDR2 and L-CDR3, or variants thereof.

[0116] In preferred embodiments, said variants have at least 80%, preferably 85, 90, 95, 97, 98 % sequence identity with the parent sequence (SEQ ID NO: 15, 16 and 17, or SEQ ID NO: 18, 19 and 20 respectively), and / or have a substitution of one, two or three amino acids with said parent sequence.

[0117] L-CDR1 is (i) SASSFVTYMF (SEQ ID NO: 15) or (ii) SANSYVSHVY (SEQ ID NO: 18);

[0118] L-CDR2 is (i) LTSNLAS (SEQ ID NO: 16) or (ii) LTSDLAS (SEQ ID NO: 19), respectively;

[0119] L-CDR3 is (i) QQWTSTPYT (SEQ ID NO: 17) or (ii) QQWSSNPLT (SEQ ID NO: 20), respectively.

[0120] In a particular embodiment, the antigen binding fragment comprises the L-CDR1 of SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16 and L-CDR3 of SEQ ID NO: 17. In another particular embodiment, the antigen binding fragment comprises the L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO:20.

[0121] In a particular embodiment, the antigen binding fragment comprises a variable light chain region comprising L-CDR1, L-CDR2 and L-CDR3 as described above or variants thereof, and further comprising framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NOs: 21-26 and SEQ ID NO:28.

[0122] In a particular embodiment, the antigen binding fragment comprises framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions QIVLTQSPALMSASPGEKVTMTC (SEQ ID NO: 21), WYQQKPRSSPKPWIY (SEQ ID NO: 22), GVPARFSGSGSGTSYSLTISSMEAEDAATYYC (SEQ ID NO: 23), and FGGGTKLEIK (SEQ ID NO: 24).

[0123] Preferably, the antigen binding fragment comprises framework regions consisting of QIVLTQSPALMSASPGEKVTMTC (SEQ ID NO: 21), WYQQKPRSSPKPWIY (SEQ ID NO: 22), GVPARFSGSGSGTSYSLTISSMEAEDAATYYC (SEQ ID NO: 23), and FGGGTKLEIK (SEQ ID NO: 24).

[0124] In another particular embodiment, the antigen binding fragment comprises framework regions that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions QIVLTQSPALIMASPGEKVTMTC (SEQ ID NO: 25), WYQQKPNSSPKPWIS (SEQ ID NO: 26), GVPARFSGSGSGTSYSLTISSMEAEDAATYYC (SEQ ID NO: 23), and FGAGTKLELK (SEQ ID NO: 28). Preferably, the antigen binding fragment comprises framework regions consisting of QIVLTQSPALIMASPGEKVTMTC (SEQ ID NO: 25), WYQQKPNSSPKPWIS (SEQ ID NO: 26), GVPARFSGSGSGTSYSLTISSMEAEDAATYYC (SEQ ID NO: 23), and FGAGTKLELK (SEQ ID NO: 28).

[0125] In a preferred embodiment, the antigen binding fragment comprises or consists of a light chain variable region sequence as set forth in :

[0126] OIVLTOSPALMSASPGEKVTMTCSASSFVTYMFWYQQKPRSSPKPWIYLTSNLASGV PARFSGSGSGTSYSLTISSMEAEDAATYYCOQWTSTPYTFGGGTKLEIK (SEQ ID NO: 29) or QIVLTOSPALIMASPGEKVTMTCSANSYVSHVYWYQQKPNSSPKPWISLTSDLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 30), wherein underlined and bold sequences represent the three CDR sequences.

[0127] In a particular embodiment, the antigen binding fragment comprises or consists of a light chain variable region sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:29 or SEQ ID NO:30. In a particular embodiment, the amino acid modification(s) with respect to SEQ ID NO:29 or SEQ ID NO:30 are not present in the CDR sequences.

[0128] In a particular embodiment, the antigen binding fragment comprises : a variable heavy chain region comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2 and H-CDR3 of SEQ ID NO: 3, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 1-3 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 1-3 ; a variable light chain region comprising L-CDR1 of SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16 and L-CDR3 of SEQ ID NO: 17, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 15-17 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 15-17.

[0129] In a particular embodiment, the antigen binding fragment comprises : a variable heavy chain region comprising H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2 and H-CDR3 of SEQ ID NO: 3, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 1-3 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 1-3 ; and further comprising framework regions of SEQ ID NOs: 7, 8, 9 and 12 or that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NOs: 7, 8, 9 and 12 ; and a variable light chain region comprising L-CDR1 of SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16 and L-CDR3 of SEQ ID NO: 17, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 15-17 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 15-17 ; and further comprising framework regions of SEQ ID NOs : 21-24 or that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NOs : 21-24.

[0130] In a particular embodiment, the antigen binding fragment comprises : a heavy chain variable region sequence as set forth in SEQ ID NO: 13, or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, or 98% sequence identity with SEQ ID NOs: 13 ; and a light chain variable region sequence as set forth in SEQ ID NO: 29, or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, or 98% sequence identity with SEQ ID NOs: 29.

[0131] In a particular embodiment, the antigen binding fragment binds to a polypeptide comprising Va7.2, wherein said antigen binding fragment binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66.

[0132] According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66, comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 or 31-35. According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66, comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13.

[0133] According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66, further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 29 or 36-39. According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66, further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 29. According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, G53, L54, F63, S65, Y72, S62, E55, E56 and R66, comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 and further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 29.

[0134] In another particular embodiment, the antigen binding fragment comprises : a variable heavy chain region comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 4-6 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 4-6 ; a variable light chain region comprising L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 18-20 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 18-20.

[0135] In particular embodiment, the antigen binding fragment comprises : a variable heavy chain region comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 4-6 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 4-6 ; and further comprising framework regions of SEQ ID NOs: 10, 8, 11 and 12 or that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NOs: 10, 8, 11 and 12 ; and a variable light chain region comprising L-CDR1 of SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20, or variants thereof, said variants having at least 80%, 85%, 90%, 95% or 98% sequence identity with SEQ ID NOs: 18-20 and / or having a substitution of one, two or three amino acids with respect to said SEQ ID NOs: 18-20, and further comprising framework regions of SEQ ID NOs: 25, 26, 23 and 28 or that show at least 80% preferably 85, 90, 95, 97, 98% sequence identity with the framework regions of SEQ ID NOs: 25, 26, 23 and 28. In a particular embodiment, the antigen binding fragment comprises : a heavy chain variable region sequence as set forth in SEQ ID NO: 14, or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, or 98% sequence identity with SEQ ID NO: 14 ; and a light chain variable region sequence as set forth in SEQ ID NO: 30, or a variant thereof having at least 80%, 85%, 90%, 95%, 97%, or 98% sequence identity with SEQ ID NO: 30.

[0136] In a particular embodiment, the antigen binding fragment binds to a polypeptide comprising Va7.2, wherein said antigen binding fragment binds at least to the following residues of Va7.2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66.

[0137] According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66, comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 14, 40-45 or 89. According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66 comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 14.

[0138] According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66, further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 30, 46-50 or 90. According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Na .2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66, further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 30.

[0139] According to a particular embodiment, said antigen biding fragment, which binds at least to the following residues of Va7.2 : L51, D52, K68, G53, L54, F63, S62, E55 and R66, comprises a heavy chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 14 and further comprises a light chain variable region having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 30.

[0140] In a particular embodiment, the variable heavy chain and variable light chain are part of the same polypeptide. In an embodiment, the antigen-binding fragment comprising a variable heavy chain and a variable light chain as described herein is a single-chain variable fragment (scFv).

[0141] In a more preferred embodiment, the antigen-binding fragment is humanized.

[0142] In a particular embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 40, 41, 42, 43, 44, 45 and 89. In a particular embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34, 35, 40, 41, 42, 43, 44,45 and 89. In a particular embodiment, the variable heavy chain of the humanized antigenbinding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34, and 35. In another particular embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 40, 41, 42, 43, 44, 45 and 89.

[0143] In a particular embodiment, the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 36, 37, 38, 39, 46, 47, 48, 49, 50 and 90. In a particular embodiment, the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NOs: 36, 37, 38, 39, 46, 47, 48, 49, 50 and 90. In a particular embodiment, the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 36, 37, 38 and 39. In another particular embodiment, the variable light chain of the humanized antigen-binding fragment 1 comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to a sequence selected in the group consisting of SEQ ID NOs: 46, 47, 48, 49, 90.

[0144] In an embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 31, 32, 33, 34 or 35 and the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 36, 37, 38, or 39. In a particular embodiment, the variable heavy chain of the humanized antigenbinding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34 and 35, and the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NO: 36, 37, 38, and 39.

[0145] In another embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 40, 41, 42, 43, 44, 45 or 89 and the variable light chain of the humanized antigen-binding fragment comprises a sequence having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 46, 47, 48, 49, 50 or 90. In a particular embodiment, the variable heavy chain of the humanized antigen-binding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NOs: 40, 41, 42, 43, 44, 45 and 89, and the variable light chain of the humanized antigen-binding fragment comprises or consists of a sequence selected in the group consisting of SEQ ID NO: 46, 47, 48, 49, 50 and 90.

[0146] In an embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment have at least 80% identity with respect to H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO:2 and H-CDR3 of SEQ ID NO:3 and / or have a substitution of one, two or three amino acids with respect to H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO:2 and H-CDR3 of SEQ ID NO:3 respectively. In an embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment comprises or consist of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80 and H-CDR3 of SEQ ID NO: 3. The humanized antigen-binding fragment may further comprise a light chain, whose CDRs have at least 80% identity with respect to L-CDR1 of SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16 and L-CDR3 of SEQ ID NO: 17, and / or have a substitution of one, two or three amino acids with respect to L-CDR1 of SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16 and L-CDR3 of SEQ ID NO: 17. In an embodiment, the CDRs of the light chain of the humanized antigen-binding fragment comprise or consist of L-CDR1 of SEQ ID NO: 15 or SEQ ID NO: 85, L-CDR2 of SEQ ID NO: 16 or SEQ ID NO: 86, and L-CDR3 of SEQ ID NO: 17. In a particular embodiment, the CDRs of the light chain of the humanized antigen-binding fragment comprise or consist of L- CDR1 of SEQ ID NO: 85, L-CDR2 of SEQ ID NO: 86, and L-CDR3 of SEQ ID NO: 17.

[0147] In another embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment have at least 80% identity with respect to H-CDR1 of SEQ ID NO:4, H-CDR2 of SEQ ID NO:5 and H-CDR3 of SEQ ID NO:6 and / or have a substitution of one, two or three amino acids with respect to H-CDR1 of SEQ ID NO:4, H-CDR2 of SEQ ID NO:5 and H-CDR3 of SEQ ID NO:6 respectively. In an embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment comprise or consist of H-CDR1 of SEQ ID NO: 4 or SEQ ID NO: 81, H-CDR2 of SEQ ID NO: 5, SEQ ID NO: 82, SEQ ID NO: 83 or SEQ ID NO: 84 or SEQ ID NO: 91, and H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92. In an embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment comprise or consist of H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 91, and H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92. In another embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment comprise or consist of H-CDR1 of SEQ ID NO: 81, H-CDR2 of SEQ ID NO: 84, and H-CDR3 of SEQ ID NO: 6. In another embodiment, the CDRs of the heavy chain of the humanized antigen-binding fragment comprise or consist of H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 or 91, and H- CDR3 of SEQ ID NO: 6 or 92. The humanized antigen-binding fragment may further comprise a light chain, whose CDRs have at least 80% identity with respect to L-CDR1 of SEQ ID NO: 18, SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20 or SEQ ID NO: 93, and / or have a substitution of one, two or three amino acids with respect to L-CDR1 of SEQ ID NOs: 18 and 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NOs: 20 and 93. In an embodiment, the CDRs of the light chain of the humanized antigen-binding fragment comprise or consist of L-CDR1 of SEQ ID NO: 18 or SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20. In an embodiment, the CDRs of the light chain of the humanized antigen-binding fragment comprise or consist of L-CDR1 of SEQ ID NO: SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20 or SEQ ID NO:93. In a particular embodiment, the heavy chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0148] H-CDR1 of SEQ ID NO: 4, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 4,

[0149] H-CDR2 of SEQ ID NO: 5, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 5, such as the variant of SEQ ID NO: 91; and

[0150] H-CDR3 of SEQ ID NO: 6, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 6, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6, such as the variant of SEQ ID NO: 92 ; and / or wherein the light chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0151] L-CDR1 of SEQ ID NO: 18, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 18, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 18, such as the variant of SEQ ID NO: 87 ;

[0152] L-CDR2 of SEQ ID NO: 19, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 19, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19 ; and

[0153] L-CDR3 of SEQ ID NO: 20, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 20, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 20, such as the variant of SEQ ID NO: 93.

[0154] In a particular embodiment, the heavy chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0155] - H-CDR1 of SEQ ID NO: 4,

[0156] - H-CDR2 of SEQ ID NO: 5 or SEQ ID NO: 91; and

[0157] - H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92 ; and / or wherein the light chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0158] - L-CDR1 of SEQ ID NO: 18 or SEQ ID NO: 87 ;

[0159] - L-CDR2 of SEQ ID NO: 19 ; and

[0160] - L-CDR3 of SEQ ID NO: 20 or SEQ ID NO: 93.

[0161] In a particular embodiment, the heavy chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0162] - H-CDR1 of SEQ ID NO: 4,

[0163] - H-CDR2 of SEQ ID NO: 5 or SEQ ID NO: 91; and

[0164] - H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92 ; and / or wherein the light chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0165] - L-CDR1 of SEQ ID NO: 87 ;

[0166] - L-CDR2 of SEQ ID NO: 19 ; and

[0167] - L-CDR3 of SEQ ID NO: 20 or SEQ ID NO: 93.

[0168] In a particular embodiment, the antigen binding fragment, which is preferably a humanized antigen binding fragment, comprises the following heavy chain CDRs :

[0169] H-CDR1 of SEQ ID NO: 4 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 4 ;

[0170] H-CDR2 of SEQ ID NO: 5 or SEQ ID NO: 91 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 5 or 91; and

[0171] H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 92 ; and / or comprises the following light chain CDRs :

[0172] L-CDR1 of SEQ ID NO: 18 or SEQ ID NO: 87 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 18 or 87;

[0173] L-CDR2 of SEQ ID NO: 19 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19; and

[0174] L-CDR3 of SEQ ID NO: 20 or SEQ ID NO: 93 or a variant thereof having a substitution of one, two or three amino acids with respect to SEQ ID NO: 20 or 93, wherein, preferably, said humanized antigen binding fragment comprises a variable heavy chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 43 and 89, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 43 and 89; and / or a variable light chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 49 and 90, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 49 and 90.

[0175] In a particular embodiment, the heavy chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0176] H-CDR1 of SEQ ID NO: 4, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 4,

[0177] H-CDR2 of SEQ ID NO: 5, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 5; and

[0178] H-CDR3 of SEQ ID NO: 6, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 6, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6 ; and / or wherein the light chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0179] L-CDR1 of SEQ ID NO: 87, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 87, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 87 ;

[0180] L-CDR2 of SEQ ID NO: 19, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 19, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19 ; and

[0181] L-CDR3 of SEQ ID NO: 20, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 20, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 20.

[0182] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4-6 ; and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L- CDR3 of SEQ ID NO: 20, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 87, 19 and 20, wherein, preferably, the heavy chain of said humanized antigen binding fragment comprises the framework regions (FR1-FR4) having respectively the sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96 and SEQ ID NO: 97, or variants thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with SEQ ID NOs: 94-97; and wherein, preferably, the light chain of said humanized antigen binding fragment comprises the framework regions (FR1-FR4) having respectively the sequence of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, or variants thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with SEQ ID NOs: 98-101.

[0183] In a particular embodiment, the antigen binding fragment is humanized and comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 43 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 49, or a variant thereof having at least 80%, such as at least 81,

[0184] 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 49.

[0185] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4-6 ; and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L- CDR3 of SEQ ID NO: 20, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 87, 19 and 20, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 43 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 49, or a variant thereof having at least 80%, such as at least 81, 82,

[0186] 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 49. In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20, wherein, said antigen binding fragment preferably comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 43 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 49, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 49.

[0187] In a preferred embodiment, the antigen-binding fragment comprises a heavy chain variable region sequence comprising or consisting of :

[0188] EVOLVQSGAEVKKPGESLRISCKASGYSFTDYYMNWVROMPGKGLEWIGRINPSNG

[0189] LVTVSS (SEQ ID NO: 43), wherein underlined and bold sequences represent the three H-CDR sequences ; and / or comprises a light chain variable region sequence comprising or consisting of :

[0190] DIOLTQSPSFLSASVGDRVTITCRASSYVSYVYWYOQKPGKAPKPWISLTSDLASGVP SRFSGSGSGTEYTLTISSLOPEDFATYYCOQWSSNPLTFGOGTKVEIK (SEQ ID NO: 49), wherein underlined and bold sequences represent the three L-CDR sequences.

[0191] In another particular embodiment, the heavy chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0192] H-CDR1 of SEQ ID NO: 4, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 4,

[0193] H-CDR2 of SEQ ID NO: 91, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 91, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 91; and

[0194] H-CDR3 of SEQ ID NO: 92, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 92, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 92 ; and / or wherein the light chain of the antigen-binding fragment, which is preferably a humanized antigen-binding fragment, has the following CDRs :

[0195] L-CDR1 of SEQ ID NO: 87, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 87, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 87 ;

[0196] L-CDR2 of SEQ ID NO: 19, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 19, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19 ; and

[0197] L-CDR3 of SEQ ID NO: 93, or a variant thereof having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% identity with SEQ ID NO: 93, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 93.

[0198] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91 and H-CDR3 of SEQ ID NO: 92, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4, 91 and 92 ; and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 93, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 87, 19 and 93, wherein, preferably, the heavy chain of said humanized antigen binding fragment comprises the framework regions (FR1-FR4) having respectively the sequence of SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, or variants thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with SEQ ID NOs: 94-97; and wherein, preferably, the light chain of said humanized antigen binding fragment comprises the framework regions (FR1-FR4) having respectively the sequence of SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 100 and SEQ ID NO: 101, or variants thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% identity with SEQ ID NOs: 98, 102, 100 and 101.

[0199] In a particular embodiment, the antigen binding fragment is humanized and comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 89, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 89 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 90, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 90.

[0200] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91 and H-CDR3 of SEQ ID NO: 92, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 4, 91 and 92 ; and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 93, or variants thereof having at least 80%, 85%, 90%, 92%, 95%, 96, 97%, 98% or 99% sequence identity to SEQ ID NOs: 87, 19 and 93, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 89, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 89 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 90, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 90.

[0201] In a particular embodiment, the antigen binding fragment is humanized and comprises : H- CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91 and H-CDR3 of SEQ ID NO: 92, and / or L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 93, wherein, said antigen binding fragment preferably comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 89, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 89 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 90, or a variant thereof having at least 80%, such as at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 90.

[0202] In a preferred embodiment, the antigen-binding fragment comprises a heavy chain variable region sequence comprising or consisting of :

[0203] EVOLVQSGAEVKKPGESLRISCKASGYSFTDYYMNWVROMPGKGLEWIGRINPSSG

[0204] LVTVSS (SEQ ID NO: 89), wherein underlined and bold sequences represent the three H-CDR sequences ; and / or comprises a light chain variable region sequence comprising or consisting of :

[0205] DIOLTOSPSFLSASVGDRVTITCRASSYVSYVYWYOQKPGKAPKPFISLTSDLASGVPS RFSGSGSGTEYTLTISSLOPEDFATYYCOQYSSNPLTFGOGTKVEIK (SEQ ID NO: 90), wherein underlined and bold sequences represent the three L-CDR sequences.

[0206] Particularly preferred antibodies are described hereafter :

[0207] “mAbl7” antibody:

[0208] - VH sequence of SEQ ID NO: 13, comprising the following CDR sequences : H-CDR1 of

[0209] SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, and the following framework sequences : FR1 of SEQ ID NO: 7, FR2 of SEQ ID NO: 8, FR3 of SEQ ID NO: 9 and FR4 of SEQ ID NO: 12.

[0210] - VL sequence of SEQ ID NO: 29, comprising the following CDR sequences : L-CDR1 of

[0211] SEQ ID NO: 15, L-CDR2 of SEQ ID NO: 16, L-CDR3 of SEQ ID NO: 17, and the following framework sequences : FR1 of SEQ ID NO: 21, FR2 of SEQ ID NO: 22, FR3 of SEQ ID NO: 23 and FR4 of SEQ ID NO: 24.

[0212] “mAbl8” antibody :

[0213] - VH sequence of SEQ ID NO: 14, comprising the following CDR sequences : H-CDR1 of

[0214] SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5, H-CDR3 of SEQ ID NO: 6, and the following framework sequences : FR1 of SEQ ID NO: 10, FR2 of SEQ ID NO: 8, FR3 of SEQ ID NO: 11 and FR4 of SEQ ID NO: 12.

[0215] - VL Sequence of SEQ ID NO: 30, comprising the following CDR sequences : L-CDR1 of

[0216] SEQ ID NO: 18, L-CDR2 of SEQ ID NO: 19, L-CDR3 of SEQ ID NO: 20, and the following framework sequences : FR1 of SEQ ID NO: 25, FR2 of SEQ ID NO: 26, FR3 of SEQ ID NO: 23 and FR4 of SEQ ID NO: 28.

[0217] “mAbl8 Variant 1”:

[0218] - VH sequence of SEQ ID NO: 43, comprising the following CDR sequences : H-CDR1 of

[0219] SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5, H-CDR3 of SEQ ID NO: 6, and the following framework sequences : FR1 of SEQ ID NO: 94, FR2 of SEQ ID NO: 95, FR3 of SEQ ID NO: 96 and FR4 of SEQ ID NO: 97.

[0220] - VL sequence of SEQ ID NO: 49, comprising the following CDR sequences : L-CDR1 of

[0221] SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19, L-CDR3 of SEQ ID NO: 20, and the following framework sequences : FR1 of SEQ ID NO: 98, FR2 of SEQ ID NO: 99, FR3 of SEQ ID NO: 100 and FR4 of SEQ ID NO: 101.

[0222] “mAbl8 variant 2” :

[0223] - VH sequence of SEQ ID NO: 89, comprising the following CDR sequences : H-CDR1 of

[0224] SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91, H-CDR3 of SEQ ID NO: 92, and the following framework sequences : FR1 of SEQ ID NO: 94, FR2 of SEQ ID NO: 95, FR3 of SEQ ID NO: 96 and FR4 of SEQ ID NO: 97.

[0225] - VL sequence of SEQ ID NO: 90, comprising the following CDR sequences : L-CDR1 of

[0226] SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19, L-CDR3 of SEQ ID NO: 93, and the following framework sequences : FR1 of SEQ ID NO: 98, FR2 of SEQ ID NO: 102, FR3 of SEQ ID NO: 100 and FR4 of SEQ ID NO: 101.

[0227] It is also provided a protein construct that comprises an anti-Va7.2 antibody moiety comprising an antigen-binding fragment such as a single domain antibody or an antigen-binding fragment that comprises both a heavy chain and a light chain as described above.

[0228] In a particular embodiment, the protein construct is a Single-chain Fv (or scFv) antibody binding fragment comprising the heavy chain variable region (VH) and light chain variable region (VL) as described above, wherein the VH / VL are present in a single polypeptide chain.

[0229] In a particular embodiment, the construct comprises two or more, e.g. three or four, antigen binding fragments as defined herein. Linear configurations are preferred, but ramified configurations may also be encompassed.

[0230] In a preferred embodiment, the protein construct comprises, or consists in, a linear repetition of at least two of said antigen binding fragments, preferably a tandem of the two same antigen binding fragments, if desired in a head to tail tandem format, i.e. Nb lCterm - Nterm Nb2 Germ.

[0231] In some embodiments, the construct may comprise one or more parts, fragments or domains of conventional chain antibodies (and in particular human antibodies) and / or of heavy chain antibodies. For example, the heavy variable chain as described above may be linked to a conventional, typically human, heavy constant chain CHI, while the light variable chain as described above may be linked to a conventional, typically human, constant light chain CL. In that case, the construct forms a Fab fragment (for Fragment antigen binding) or Fab domain. Assembly of Fab domains is accomplished via natural pairing of light and heavy chains without the use of peptide linkers. Mutations at the interface of light and heavy constant chains of the Fab fragments or domains may be introduced in order to maximize propensity of cognate pairing between light and heavy chains. In a naturally occurring antibody, there are two types of light chain, lambda (X) and kappa (K), that contain both the light constant region and the light variable region. The constant light chain of the construct of this invention may be derived from a lambda or kappa light chain, preferably from a kappa light chain.

[0232] If effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADP) are desired, the protein construct of the invention can further comprise a Fc (Fragment crystallisable) domain providing these effector functions.

[0233] In another embodiment, the constant region includes a CH2 domain and at least a portion of a hinge region. The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgGl, IgG2, IgG3, IgG4, or other suitable classes, mutated or not. More preferably the hinge region is derived from a human IgGl heavy chain. In one embodiment, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype.

[0234] In other embodiments, the construct comprises the antigen binding fragment, typically the single domain antibody or the Fab fragment, that is linked to an immunoglobulin or a portion or fragment thereof. For example, the polypeptide, or fusion protein comprises the antigen binding fragment that is linked to an Fc domain, notably a human Fc region. In a preferred embodiment, the Fc region comprises the dimerized CH2 and CH3 domains of an immunoglobulin. The Fc domain may be introduced in the construct if effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADP) are desired, in which case the choice of the Fc domain will depend on the type of desired effector functions, or to improve antibody properties such as in vivo half-life.

[0235] In a particular embodiment, the protein construct of the invention has an immunoglobulin-like structure, comprising: - two identical antigen-binding arms, each comprising the heavy variable chain, light variable chain, CHI and CL domains as defined above, more preferably each consisting in a Fab fragment comprising the antigen-binding fragment of the invention as defined above;

[0236] - the dimerized CH2 and CH3 domains of an immunoglobulin;

[0237] - the hinge region of an IgA, IgG, or IgD, linking the C-terminal ends of the CHI domains of the antigen-binding arms to the N-terminal ends of the CH2 domains.

[0238] Preferably, the CH2 and CH3 domains and / or the hinge region are derived from a same immunoglobulin or from immunoglobulins of the same isotype and subclass as the CHI domains of the antigen-binding arm.

[0239] In a particular embodiment, the protein construct having an immunoglobulin-like structure is humanized; in a particular embodiment, it is a humanized antibody.

[0240] The CH2 and CH3 as well as the hinge regions from native immunoglobulins can be used. It is also possible to mutate them, if desired, for instance in order to modulate the effector function of the antibody or the in vivo half-life of the antibody. In some instances, whole or part of the CH2 or the CH3 domain can be omitted.

[0241] The alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc fusion protein (PCT publication WO 01 / 58957). Accordingly, the junction region of a protein or polypeptide of the present invention can contain alterations that increase serum half-life and that, relative to the naturally occurring sequences of an immunoglobulin heavy chain, preferably lie within about 10 amino acids of the junction point.

[0242] In certain embodiments, an antibody may be altered to increase, decrease or eliminate the extent to which it is glycosylated. Glycosylation of polypeptides is typically either "N-linked" or "O- linked."

[0243] "N-linked" glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. "O-linked" glycosylation refers to the attachment of one of the sugars N- acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxy lysine may also be used. Addition or deletion of N-linked glycosylation sites to the antibody may be accomplished by altering the amino acid sequence such that one or more of the above- described tripeptide sequences is created or removed. Addition or deletion of O-linked glycosylation sites may be accomplished by addition, deletion, or substitution of one or more serine or threonine residues in or to (as the case may be) the sequence of an antibody.

[0244] The antigen-binding fragments or protein constructs as described above can be useful for the detection and purification of MAIT cells or more generally cells displaying the Va7.2 chain. For example, conjugation or covalent binding of the antigen-binding fragment or protein construct to a detectable moiety, such as a marker or label, allows for the detection, enumeration and / or purification of MAIT cells or cells displaying the Va7.2 chain, or depletion or modulation of the activity of MAIT cells or cells displaying the Va7.2 chain.

[0245] Sorting and / or purification of MAIT cells or cells displaying the Va7.2 chain may be performed by contacting the sample with any antibodies, antigen-binding fragments or protein constructs of the invention under conditions that allow the cells to bind to the antibody, wherein the antibody is conjugated or covalently bound to a solid support, then eluting the bound cells from the solid support-bound antibody. Alternatively, sorting and / or purification may be performed by providing a fluorescently-labeled anti-Va7.2 antibody, antigen-binding fragment or protein construct of the present invention and a biological sample, then using the labeled antibody to purify MAIT cells or cells displaying Va7.2 from the biological sample by Fluorescence-Activated Cell Sorting (FACS).

[0246] Markers of interest, used in combination with an antigen-binding fragment or protein construct of the invention, include but are not limited to : 2B4 (CD244), CD161, CD137, CD25, CD26, CD28, CD94, CD96, IL12Rb (CD212), IL18Ra (CD218b), NKG2D (CD314), CD122, CD127, CD27 and CD45RO. Preferably, methods for detecting, depleting, purifying, or modulating the activity of MAIT cells or cells expressing the Va7.2 chain, in vitro or in vivo, comprise the use of two, three, four, five or more agents selective for a polypeptide selected from the group 2B4 (CD244), CD161, CD137, CD25, CD26, CD28, CD94, CD96, IL12Rb (CD212), IL18Ra (CD218b), NKG2D (CD314), CD122, CD127, CD27 and CD45RO. Detection of MAIT cells or cells expressing the Va7.2 chain using this method may be performed using a flow cytometry device or microarrays.

[0247] Conjugation to a detectable moiety is useful, inter alia, when an antibody of the invention is used for diagnostic purposes. Such purposes include, but are not limited to, assaying biological samples, e.g., a blood sample or mucosal tissue biopsy, for the presence of MAIT cells, and detecting the presence, level, or activity of MAIT cells in an individual.

[0248] Such methods are useful, e.g., for diagnosing conditions caused by or associated with an increase in MAIT cell activity or number, e.g. cancer, infection, irritable bowel syndrome, Crohn's disease, celiac disease or ulcerative colitis. Labeled antibodies of the invention can also be used in FACS sorting to purify or isolate MAIT cells from a biological sample.

[0249] In another embodiment, antibodies, antigen-binding fragments or protein constructs of the invention may be used to block the interaction between the protein MR1 and the Va7.2 chain, preferably the Va7.2-Ja33 chain.

[0250] An antibody, antigen-binding fragment or protein construct of the invention may be used to activate MAIT cells, and in particular they can be administered to activate MAIT cells or increase the activity of MAIT cells to a patient in need thereof.

[0251] Antibodies, antigen-binding fragments or protein constructs of the invention can be administered to a non-human primate model of the disorder in order to assess the ability of the antibody to prevent or ameliorate the disorder, or a symptom thereof, or to modulate the activity of MAIT cells in the primate. In a particular embodiment of the invention, the antibodies, antigen-binding fragments or protein constructs are used for preclinical studies on model species, preferably on the murine model (Mus musculus) or the cynomolgus monkey (Macaca fascicularis), more preferably on the cynomolgus monkey. In a preferred embodiment, the antibodies, antigen-binding fragments or protein constructs of the invention display crossreactivity to the Va7.2 TCR of the cynomolgus monkey, with binding to the Va7.2 TCR of the cynomolgus monkey representing at least 50%, preferably at least 60 %, 70%, 80%, 90% of binding to the human Va7.2 TCR. In an embodiment, the antibodies, antigen-binding fragments or protein constructs of the invention are used for the identification, selection or purification of MAIT cells in non-human species, in particular NHP model species, and preferably in the cynomolgus monkey.

[0252] Protein constructs

[0253] The invention further provides protein constructs which comprise at least one anti-Va7.2 binding fragment as defined herein, optionally linked to at least another polypeptide.

[0254] The protein construct may be monovalent or multivalent.

[0255] The term “multivalent” herein refers a construct having multiple antigen-binding sites. A multivalent construct may comprise identical or different antigen-binding sites.

[0256] The protein construct may also be monospecific or multispecific.

[0257] The term “multispecific” herein refers to the ability of the construct to simultaneously bind to two or more different antigens or two or more different epitopes of the same antigen (i.e. it is a multiparatopic construct). In a particular embodiment, the construct is a bispecific or trispecific antibody. In a particular embodiment, the construct is a biparatopic antibody.

[0258] In a particular embodiment, the protein construct is multispecific, preferably bispecific, and at least comprises the anti-Va7.2 antigen-binding fragment of the invention and another fragment binding to another antigen, such as a tumor associated antigen (TAA).

[0259] In a particular embodiment, the protein construct is multispecific, preferably bispecific, and comprises at least one domain that binds a TAA.

[0260] Particular examples of such TAAs include CD19, EGFR and HER2. Preferably, the TAA is HER2.

[0261] Generally speaking, any person skilled in the art knows how to produce antibodies that specifically bind to any of said TAAs. Many are commercialized.

[0262] More preferably, the multispecific protein construct described herein comprises tandemly arranged Fab fragments, in any order, one comprising the anti-Va7.2 -binding fragment of this invention, the other comprising at least one domain that binds a TAA. In a particular embodiment, the C-terminal end of the CHI domain of a first Fab fragment is linked to the N- terminal end of the VH domain of the following Fab fragment through a polypeptide linker. In a particular embodiment, the construct of the invention is a multispecific antigen-binding fragment Fab-Fab, which does not contain an Fc domain.

[0263] In a particular embodiment, the multispecific protein construct may comprise the anti-Va7.2 antigen-binding fragment of the invention and a T-cell receptor that binds a specific antigen.

[0264] In a particular embodiment, the multispecific construct of the invention may also comprise another antigen-binding fragment that binds to a molecule displayed at the surface of MAIT cells, this molecule not being Va7.2.

[0265] Design of particular multispecific antibodies

[0266] It is herein provided multispecific antigen-binding fragment(s) and multispecific antibody constructs, comprising said fragments, wherein each multispecific antigen-binding fragment consists essentially of tandemly arranged Fab fragments.

[0267] In a particular embodiment, the multispecific antigen-binding consists essentially of tandemly arranged Fab fragments (Fab-Fab), wherein at least one Fab fragment comprises the anti-Va7.2 -binding fragment of the invention. In a particular embodiment, the multispecific antigenbinding consists essentially of two tandemly arranged Fab fragments (Fab-Fab), wherein one Fab fragment comprises the anti-Va7.2-binding fragment of the invention, and wherein the other Fab fragment comprises a domain that binds a TAA.

[0268] Such fragments and constructs preferably comprise chains from human immunoglobulins, preferably IgG, still preferably IgGl.

[0269] In case of a multispecific antigen-binding fragment comprising more than two different Fab fragments, the polypeptide linkers separating the Fab fragments can be identical or different.

[0270] According to a preferred embodiment, it is provided a multispecific antibody that comprises two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined above. The antigen-binding arms can be linked together in diverse ways. If one wishes to obtain an antibody without Fc-mediated effects or an antibody monovalent for each of the two antigens it targets, the antibody will comprise no Fc region. In this case, the two antigen-binding arms can be linked together for instance:

[0271] - by homodimerization of the antigen-binding arms through the inter-chain disulfide bonds provided by the polypeptide linker(s) separating the Fab fragments; and / or

[0272] - through the addition at the C-terminal end of each antigen-binding arm, of a polypeptide extension containing cysteine residues allowing the formation of inter-chain disulfide bonds, and homodimerization of said polypeptide extension resulting in a hinge-like structure; by way of non-limiting examples, said polypeptide extension may be for instance a hinge sequence of an IgGl, IgG2 or IgG3;

[0273] - through a linker, preferably a semi-rigid linker, joining the C-terminal ends of the heavy chains of the two antigen-binding arms to form a single polypeptide chain and maintaining said antigen-binding arms at a sufficient distance between each other.

[0274] Alternatively, if effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADP) or bivalent binding for each of the two antigens are desired, a multispecific antibody of the invention can further comprise a Fc domain providing these effector functions. The choice of the Fc domain will depend on the type of desired effector functions.

[0275] In this case, a multispecific antibody of the invention has an immunoglobulin-like structure, comprising:

[0276] - two identical multispecific antigen-binding arms as defined above;

[0277] - the dimerized CH2 and CH3 domains of an immunoglobulin;

[0278] - either the hinge region of an IgA, IgG, or IgD, linking the C-terminal ends of the CHI domains of the antigen-binding arms to the N-terminal ends of the CH2 domains, or alternatively, when the CH4 domains that follow the CH3 domains come from an IgM or IgE, the C-terminal ends of the CHI domains of the antigen-binding arms in this case can be linked directly to the N- terminal ends of the CH2 domains.

[0279] Preferably, the CH2 and CH3 domains, the hinge region and / or the CH4 domains are derived from a same immunoglobulin or from immunoglobulins of the same isotype and subclass as the CHI domains of the antigen-binding arm. The CH2, CH3, and optionally CH4 domains, as well as the hinge regions from native immunoglobulins can be used. It is also possible to mutate them, if desired, for instance in order to modulate the effector function of the antibody. In some instances, whole or part of the CH2 or the CH3 domain can be omitted.

[0280] The invention more particularly provides bispecific tetravalent antibodies, comprising two binding sites to each of their targets, and a functional Fc domain allowing the activation of effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis. Such preferred antibodies are full length antibodies. However preferred antibodies carry mutations in the Fc domain so as to avoid or reduce binding to Fc gamma receptors.

[0281] The antibodies preferably comprise heavy chains and light chains from human immunoglobulins, preferably IgG, still preferably IgGl.

[0282] The light chains may be lambda or kappa light chains; they preferably are Kappa light chains.

[0283] In a preferred embodiment, a linker links IgG Fab domains in a tetra-Fab bispecific antibody format, the amino acid sequence of which comprises the heavy chain sequences of at least two Fab domains joined by said polypeptide linker, followed by the native hinge sequence, followed by the IgG Fc sequence, co-expressed with the appropriate IgG light chain sequences.

[0284] A preferred example of the bispecific antibodies of the invention, named BiXAb antibodies, which have an IgG-like structure, is illustrated in Figure 1.

[0285] In a particular embodiment, the bispecific antibodies of the invention comprise

[0286] - a continuous heavy chain constructed of an Fc (Hinge-CH2-CH3)

[0287] - followed by antibody 1 Fab heavy chain (CH1-VH) and the successive Fab heavy chain (CH1- VH) of antibody 2, the latter joined by a polypeptide linker sequence, e.g. a linker as described in greater details below,

[0288] - and during protein expression the resulting heavy chain assembles into dimers while the coexpressed antibody 1 and antibody 2 light chains (VL-CL) associate with their cognate heavy chains to form the final tandem F(ab)’2-Fc molecule, the antibody 1 (Abl) and the antibody 2 (Ab2) being different, and wherein one of Abl or Ab2 comprises the anti-Va7.2 binding fragment of the invention.

[0289] In a preferred embodiment, described are bispecific antibodies, which comprise

[0290] • two Fab fragments with different CHI and CL domains consisting of a) Fab fragment having CHI and C-Kappa domains derived from a human IgGl / Kappa, and the VH and VL domains of Abl, b) Fab fragment having CHI and C-Kappa domains derived from a human IgGl / Kappa and the VH and VL domains of Ab2, the Fab fragments being tandemly arranged in the following order the C-terminal end of the CHI domain of Abl Fab fragment being linked to the N- terminal end of the VH domain of Ab2 Fab fragment through a polypeptide linker, the hinge region of a human IgGl linking the C-terminal ends of CHI domain of Ab2 fragment to the N-terminal of the CH2 domain, the dimerized CH2 and CH3 domains of a human IgGl, preferably with one or several mutations that reduce or eliminate the interaction with Fc gamma receptors ; and wherein one of Abl or Ab2 comprises the anti-Va7.2 binding fragment of the invention.

[0291] According to the invention, Abl and Ab2 are, independently, an antibody that specifically binds Va7.2 (such as those described in greater details above), and an antibody that specifically binds to another antigen (such as a tumor associated antigen), or vice versa.

[0292] A preferred construct of the invention is a multispecific antigen-binding fragment Fab-Fab, which does not contain the Fc domain.

[0293] Such Fab-Fab constructs typically comprise two different Fab domains. They possess the same Light Chains as in the corresponding BiXAb antibodies; however, the Heavy Chain of Fab- Fabs is shortened in such a fashion so that their most C-terminal residue is Cysteine-220 (in EU numbering).

[0294] The assembly of Fab domains is accomplished via natural pairing of Light and Heavy chains without the use of peptide linkers.

[0295] In order to maximize propensity of cognate pairing between Light and Heavy chains, one may contemplate introducing mutations at the interface of Light and Heavy chains (CL / CH1 interface) in Fab fragments. In preferred embodiments, each CHI domain carries at least one mutation, and each CL1 domain also carries at least one mutation, which mutations are selected so that a correct cognate pairing of the CHI and CL1 domains is improved.

[0296] These mutations can be selected from the following list:

[0297] - de novo-introduced ionic pairs or reversed polarity charged mutations of native ionic pairs already present at the interface of the Heavy and Light chains of the Fab fragment;

[0298] - “knobs-into-holes” mutations;

[0299] - mutations that resurface opposing constant regions of Heavy and Light chain interfaces in Fab fragments to change them from strongly polar to highly hydrophobic or vice versa.

[0300] Several sets of mutations are thus suitable, as described in greater details below.

[0301] Of note, throughout the present description, amino acid sequences and the sequence position numbers used herein for the CHI and CL domains are defined according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0302] Residues that can be mutated in the VL domain may be e.g. selected from the group consisting of D 1, W 36, Q 38, A 43, P 44, T 85, F 98, and Q 100 (e.g. Q100C).

[0303] Residues that can be mutated in the CL kappa domain may be e.g. selected from the group consisting of S 114, F 116, F 118, E 123 (e.g. E123K), Q 124, T 129, S 131, V 133, L 135, N 137, Q 160, S 162, S 174, S 176, T 178, and T 180.

[0304] Residues that can be mutated in the CL lambda domain may be e.g. selected from the group consisting of S 114, T 116, F 118, E 123, E 124, K 129, T 131, V 133, L 135, S 137, V 160, T 162, A 174, S 176, Y 178, and S 180.

[0305] Residues that can be mutated in the VH domain may be e.g. selected from the group consisting of V 37, Q 39, G 44 (e.g.G44C), R 62, F 100, W 103, and Q 105.

[0306] Residues that can be mutated in the CHI domain may be e.g. selected from the group consisting of L 124, A 139, L 143, D 144, K 145, D 146, H 172, F 174, P 175, Q 179, S 188, V 190, T 192, and K 221 (e.g.K221E).

[0307] Specific mutations are described in US patent applications 2014 / 0200331, 2014 / 150973, 2014 / 0154254, and international patent application W02007 / 147901, all incorporated herein by reference. In a preferred embodiment, a pair of interacting polar interface residues is exchanged for a pair of neutral and salt bridge forming residues. The replacement of Thrl92 by a glutamic acid or aspartic acid on CHI chain and exchange of Asnl37 to a Lys on CL chain can be selected, optionally with a substitution of the serine residue at position 114 of said CL domain with an alanine residue.

[0308] In another set of mutations, one can replace the Leul43 of the CHI domain by a Gin residue, while the facing residue of the CL chain, that is Val33, is replaced by a Thr residue. This first double mutation constitutes the switch from hydrophobic to polar interactions. Simultaneously a mutation of two interacting serines (Seri 88 on CHI chain and Ser 176 on CL chain) to valine residues can achieve a switch from polar to hydrophobic interactions.

[0309] In yet another embodiment, the mutations can comprise substitution of the leucine residue at position 124 of CHI domain with a glutamine and substitution of the serine residue at position 188 of CHI domain with a valine residue; and substitution of the valine residue at position 133 of CL domain with a threonine residue and substitution of the serine residue at position 176 of said CL domain with a valine residue.

[0310] The "knob into holes" mutations include a set of mutations (KH1) wherein Leu 124 and Leu 143 of the CHI domain have been respectively replaced by an Ala and a Glu residue while the Vai 33 of the CL chain has been replaced by a Trp residue, while, in the set of mutations named H2, the Vai 90 of the CHI domain has been replaced by an Ala residue, and the Leu 135 and Asnl37 of the CL chain have respectively been replaced by a Trp and an Ala residue.

[0311] The preferred mutations are disclosed below:

[0312] In a particular embodiment, the multispecific antibody may carry a double mutation, e.g. one arm with the CR3 mutation and the other with the Mut4 mutation.

[0313] In a particular embodiment, the multispecific antibody further comprises a Fc region of an immunoglobulin comprising Hinge-CH2-CH3 domains, which Fc region is linked to both antigen-binding arms by said Hinge domain, linking the C-terminal ends of CHI domains of the antigen-binding arms to the N-terminal ends of the CH2 domains.

[0314] Specific mutations at the interface in the CH3 or CH2 domains of the Fc may be contemplated to favor hetero -dimerization of two heavy chains instead of their natural homo-dimerization. Such mutations may be selected from the following list:

[0315] - de novo-introduced ionic pairs or reversed polarity charged mutations of native ionic pairs already present at the interface of two Heavy chains of the Fc domain;

[0316] - knobs-into-holes types mutations, well known and described in the art;

[0317] - mutations that resurface two opposing Heavy chain interfaces, e.g. to change them from strongly polar to highly hydrophobic, or vice versa.

[0318] Also, specific mutations in the IgGl Fc domain decreasing or eliminating binding to Fc gamma receptors may be utilized, including but not limited to:

[0319] - L234A / L235A

[0320] - N297A (eliminating N-linked glycosylation site)

[0321] - L234A / L235A / G237A / P238S / H268A / A330S / P33 IS

[0322] - Or specific combination of positional substitutions of any of the following residues: L234A, L235A, G236R, G237A, P238S, H268A, L328R, A330S, P331S (EU numbering).

[0323] In a particular embodiment, the Fc region comprises of consists of the amino acid sequence of SEQ ID NO: 88, or a functional variant thereof having at least 80%, such as 85%, 90%, 95% or at least 98% sequence identity to SEQ ID NO: 88.

[0324] Any of the molecules described herein can be modified to contain additional non-proteinaceous moieties that are known in the art and readily available, e.g., by PEGylation, hyperglycosylation, and the like. Modifications that can enhance serum half-life or stability against proteolytic degradation are of interest.

[0325] The antibodies of the invention may be glycosylated or not, or may show a variety of glycosylation profiles. In a preferred embodiment, antibodies are unglycosylated on the variable region of the heavy chains, but are glycosylated on the Fc region.

[0326] One may use humanized forms of a reference non-human antibody. In a humanization approach, complementarity determining regions (CDRs) and certain other amino acids from donor variable regions are grafted into human variable acceptor regions and then joined to human constant regions. See, e.g. Riechmann et al., Nature 332:323-327 (1988); U.S. Pat. No. 5,225,539.

[0327] Design of the linkers

[0328] In a particular embodiment, a polypeptide linker is used to link the Fab fragment that binds Va7.2 of this invention, and the Fab fragment that binds the other antigen, such as the tumor- associated antigen.

[0329] It is also designated “hinge-derived polypeptide linker sequence” or “pseudo hinge linker”, and comprises all or part of the sequence of the hinge region of one or more immunoglobulin(s) selected among IgA, IgG, and IgD, preferably of human origin. Said polypeptide linker may comprise all or part of the sequence of the hinge region of only one immunoglobulin. In this case, said immunoglobulin may belong to the same isotype and subclass as the immunoglobulin from which the adjacent CHI domain is derived, or to a different isotype or subclass. Alternatively, said polypeptide linker may comprise all or part of the sequences of hinge regions of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal portion of the polypeptide linker, which directly follows the CHI domain, preferably consists of all or part of the hinge region of an immunoglobulin belonging to the same isotype and subclass as the immunoglobulin from which said CHI domain is derived.

[0330] Optionally, said polypeptide linker may further comprise a sequence of from 2 to 15, preferably of from 5 to 10 N-terminal amino acids of the CH2 domain of an immunoglobulin.

[0331] The polypeptide linker sequence typically consists of less than 80 amino acids, preferably less than 60 amino acids, still preferably less than 40 amino acids.

[0332] In some cases, sequences from native hinge regions can be used; in other cases point mutations can be brought to these sequences, in particular the replacement of one or more cysteine residues in native IgGl, IgG2 or IgG3 hinge sequences by alanine or serine, in order to avoid unwanted intra-chain or inter-chains disulfide bonds.

[0333] In a particular embodiment, the polypeptide linker sequence comprises or consists of amino acid sequence EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 71), wherein XI, X2, X3, X4, X5, X6, X7, X8, X9, X10, identical or different, are any amino acid. In particular, the polypeptide linker sequence may comprise or consist of a sequence selected from the group consisting of EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 72); EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 73); EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 74); EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG(SEQ ID NO:75);

[0334] EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO:76) and EPKSCDKTHTAPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 77).

[0335] In a preferred embodiment, the polypeptide linker sequence comprises or consists of SEQ ID NO: 77.

[0336] In a particular embodiment, XI, X2 and X3, identical or different, are Threonine (T) or Serine (S).

[0337] In another particular embodiment, XI, X2 and X3, identical or different, are selected from the group consisting of Ala (A), Gly (G), Vai (V), Asn (N), Asp (D) and He (I), still preferably XI, X2 and X3, identical or different, may be Ala (A) or Gly (G).

[0338] Alternatively, XI, X2 and X3, identical or different, may be Leu (L), Glu (E), Gin (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gin (Q).

[0339] In a particular embodiment, X4 and X5, identical or different, are any amino acid selected from the group consisting of Serine (S), Cysteine (C), Alanine (A), and Glycine (G).

[0340] In a preferred embodiment, X4 is Alanine (A), Serine (S) or Cysteine (C). In a preferred embodiment, X4 is Alanine (A).

[0341] In a preferred aspect, X5 is Alanine (A) or Cysteine (C).

[0342] In a particular embodiment, X6, X7, X8, X9, X10, identical or different, are any amino acid other than Threonine (T) or Serine (S). Preferably X6, X7, X8, X9, X10, identical or different, are selected from the group consisting of Ala (A), Gly (G), Vai (V), Asn (N), Asp (D) and He (I).

[0343] Alternatively, X6, X7, X8, X9, X10, identical or different, may be Leu (L), Glu (E), Gin (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E), or Gin (Q).

[0344] In a preferred embodiment, X6, X7, X8, X9, X10, identical or different, are selected from the group consisting of Ala (A) and Gly (G).

[0345] In still a preferred embodiment, X6 and X7 are identical and are preferably selected from the group consisting of Ala (A) and Gly (G). In a preferred embodiment, the polypeptide linker sequence comprises or consists of sequence SEQ ID NO: 71, wherein

[0346] XI, X2 and X3, identical or different, are Threonine (T), Serine (S);

[0347] X4 is Alanine (A), Serine (S) or Cysteine (C);

[0348] X5 is Alanine (A) or Cysteine (C);

[0349] X6, X7, X8, X9, X10, identical or different, are selected from the group consisting of Ala (A) and Gly (G).

[0350] In another preferred embodiment, the polypeptide linker sequence comprises or consists of sequence SEQ ID NO: 71, wherein

[0351] XI, X2 and X3, identical or different, are Ala (A) or Gly (G);

[0352] X4 is Alanine (A), Serine (S) or Cysteine (C);

[0353] X5 is Alanine (A) or Cysteine (C);

[0354] X6, X7, X8, X9, X10, identical or different, are selected from the group consisting of Ala (A) and Gly (G).

[0355] In embodiments wherein the antibodies comprise different Fab fragments, the polypeptide linkers separating the Fab fragments can be identical or different.

[0356] Production of the antigen-binding fragments and protein constructs

[0357] Also provided herein is an isolated nucleic acid encoding the antigen-binding fragments and constructs disclosed herein. Also provided are expression vectors comprising the isolated nucleic acid molecules disclosed herein. Isolated host cells comprising the nucleic acid molecules or vectors as described herein are also provided by the invention.

[0358] The nucleic acids coding for the light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Such control sequences typically include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., a tetracycline resistance gene or a neomycin resistance gene or another gene or cassette for resistance to an antibiotic, or auxotrophic marker genes, to permit detection of those cells transformed with the desired DNA sequences. The recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virus (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0359] The recombinant vectors for expression of the antibodies described herein typically contain a nucleic acid encoding the antibody amino acid sequences operably linked to a promoter, either constitutive or inducible. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the antibody. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0360] Antigen-binding fragment, protein construct and multispecific molecule as described herein may be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, insect, and mammalian cells. It is not necessary that the recombinant antibodies of the invention be glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are human embryonic kidney line (HEK 293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells / - or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells).

[0361] It is further provided methods of producing antigen-binding fragments or protein constructs as described herein, comprising culturing the host cell so that the antigen-binding fragment or protein construct is produced, and / or recovering and / or isolating the antigen-binding fragments or protein construct from the host cell.

[0362] A host cell transformed with the nucleic acid that encodes the antigen-binding fragment as defined herein is also encompassed.

[0363] Production of the multispecific antibodies

[0364] Nucleic acids encoding heavy and light chains of the antibodies of the invention are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences.

[0365] In one example, both the heavy and light chain coding sequences (e.g., sequences encoding a VH and a VL, a VH-CH1 or a VL-CL, are included in one expression vector. In another example, each of the heavy and light chains of the antibody is cloned into an individual vector. In the latter case, the expression vectors encoding the heavy and light chains can be cotransfected into one host cell for expression of both chains, which can be assembled to form intact antibodies either in vivo or in vitro.

[0366] In a particular embodiment, a host cell is co-transfected with three independent expression vectors, such as plasmids, leading to the coproduction of all three chains (namely the heavy chain HC, and two light chains LC1 and LC2, respectively) and to the secretion of the multispecific antibody.

[0367] More especially the three vectors may be advantageously used in a following molecular ratio of 3:2:2 (HC : LC1 : LC2).

[0368] The recombinant vectors for expression of the antibodies described herein typically contain a nucleic acid encoding the antibody amino acid sequences operably linked to a promoter, either constitutive or inducible. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the antibody. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0369] Multispecific antibodies as described herein may be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, insect, and mammalian cells. It is not necessary that the recombinant antibodies of the invention be glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are human embryonic kidney line (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells / - or + DHFR (CHO, CHO-S, CHO- DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells).

[0370] Mammalian tissue cell culture is preferred to express and produce the polypeptides because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various Cos cell lines, HeEa cells, preferably myeloma cell lines, or transformed B -cells or hybridomas.

[0371] In a most preferred embodiment, the multispecific, preferably bispecific, antibodies of the invention are produced by using a CHO cell line, most advantageously a ExpiCHO cell line.

[0372] Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like.

[0373] The vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.

[0374] Host cells are transformed or transfected with the vectors (for example, by chemical transfection or electroporation methods) and cultured in conventional nutrient media (or modified as appropriate) for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be further isolated or purified to obtain preparations that are substantially homogeneous for further assays and applications. Standard protein purification methods known in the art can be used. For example, suitable purification procedures may include fractionation on immunoaffinity, Hydrophobic interaction chromatography (HIC), or ion-exchange columns, ethanol precipitation, high-performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, and gel filtration (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., 1982). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.

[0375] In vitro production allows scale-up to give large amounts of the desired multispecific, preferably bispecific, antibodies of the invention. Such methods may employ homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges.

[0376] Therapeutic applications

[0377] A further aspect of the invention is a pharmaceutical composition comprising an antigen binding fragment, a protein construct or a multispecific molecule, more particularly an antibody, as described herein. Another aspect of the invention is the use of an antigen binding fragment, protein construct or multispecific molecule, more particularly an antibody, according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antigen binding fragment, a protein construct or a multispecific molecule, more particularly an antibody, according to the invention.

[0378] In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing an antigen binding fragment, a protein construct or a multispecific molecule, more particularly an antibody as defined herein, formulated together with a pharmaceutical carrier. A composition of the present invention can be administered by a variety of methods known in the art. Any suitable route of administration is encompassed, including intravenous, oral, subcutaneous, intradermal, or mucosal administration. In another particular embodiment, an injection directly to the site of the tumor, or in its vicinity, is contemplated.

[0379] The composition of the invention that comprises a multispecific molecule that bind a tumor associated antigen is particularly useful for treating a tumor, especially a solid tumor, such as a cancer selected from the group consisting of a lung cancer (e.g. small cell lung cancer, nonsmall cell lung cancer), skin cancer, melanoma, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, uroepithelial cancers, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile ducts, uterine, adrenal, cancers, sarcomas. Hematological malignancies (e.g. lymphoma, leukemia, multiple myeloma) are encompassed as well.

[0380] Conjugates

[0381] It is further provided conjugates that comprise the antigen binding fragment, the protein construct or the multispecific molecule as described herein.

[0382] In such conjugates, the antigen binding fragment, the protein construct or the multispecific molecule are linked, preferably by chemical conjugation, to another moiety (herein also designated “accessory moiety”).

[0383] In the context of the present disclosure, a “conjugate” encompasses an antigen-binding fragment, protein construct or multispecific molecule described herein, covalently linked to an accessory moiety, e.g. an effector molecule. The effector molecule can be, for example, a drug, or any therapeutic agent, immunologic agent, detectable label, protein, nucleic acid, lipid, radioligand, radionuclide, nanoparticle, photon absorber, photosensitizer, etc. Immunologic agents are drugs that can modify the immune response. When the accessory moiety is linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an “antibody-drug conjugate” or “ADC”. As used herein, the term "radionuclide" has its general meaning in the art and refers to atoms with an unstable nucleus, characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or via internal conversion.

[0384] Methods for conjugation or covalent bonding of these accessory moieties to antibodies are well known in the art (see, e.g., WO 2014 / 160160, U.S. Patent Nos. 5,208,020 and 5,416,064; and Chari et al., 1992 Cancer Res. 52:127-131) .

[0385] The conjugates of this invention can be used for therapeutic, diagnostic or detection purposes. In particular, they can be used to activate MAIT cells or increase their activity, inhibit the activity of MAIT cells, or deplete MAIT cells.

[0386] In other embodiments, conjugates are multispecific antibodies of the invention that binds a tumor associated antigen (TAA). In a particular embodiment, they are administered for the treatment of cancer.

[0387] The present invention, thus generally described above, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the instant invention.

[0388] EXAMPLES antibodies

[0389] Generation of anti-Va7.2 antibodies

[0390] To obtain anti-Voc7.2 binders, cDNA libraries were screened using Phage display technology from 6 immunized wild type mice with the recombinant human Voc7.2V|313 TCR (human TRAVl-2-TRAJ33_VK of SEQ ID NO:63 and human TRBV6-1-TRBJ2-2 of SEQ ID NO: 64). Three rounds of selection were performed using two recombinant TCRs (human TRAV1-2- TRAJ33_VK / human TRBV6-1-TRBJ2-2 and human TRAVl-2-TRAJ33_FM / human TRBV20-1) and the irrelevant TCR (human TRAV12-TRAJ33 / TRBV6-1-TRBJ2-2) for counter selection. Finaly, 270 unique sequences were identified. 20 VH / VL sequences of interest were synthetized. After codon optimization for mammalian expression, they were cloned into pCDN3.4 vector containing the human constant IgGl heavy chain (SEQ ID NO: 55) or kappa light chain (SEQ ID NO: 56) for VH or VL sequences cloning respectively. Each plasmid was amplified in E. coli and purified by midi-prep before quality controls (absorbance A260 / A280, level of endotoxin and sequencing).

[0391] Two mAbs, herein named mAbl7 and mAbl8, were identified. The variable heavy chain of mAbl7 is shown in SEQ ID NO: 13, with its H-CDR1, H-CDR2 and H-CDR3 consisting of SEQ ID NO 1, 2 and 3, respectively. The variable heavy chain of mAbl8 is shown in SEQ ID NO: 14, with its H-CDR1, H-CDR2 and H-CDR3 consisting of SEQ ID NO 4, 5 and 6, respectively. The variable light chain of mAbl7 is shown in SEQ ID NO: 29, with its L-CDR1, L-CDR2 and L-CDR3 consisting of SEQ ID NO 15, 16 and 17, respectively. The variable light chain of mAbl8 is shown in SEQ ID NO: 30, with its L-CDR1, L-CDR2 and L-CDR3 consisting of SEQ ID NO 18, 19 and 20, respectively.

[0392] These two mAbs, mAbl7 and mAbl8, were then humanized using a CDR-grafting approach. Briefly, the essential CDR and framework residues from the original mouse antibodies sequence were identified and grafted into the variable and constant regions of germline human antibodies. After grafting of the mAbl7 CDRs, five heavy chains (SEQ ID NOs: 31-35) and four kappa light chains (SEQ ID NOs: 36-39) were generated. These chains were designed from VH1-2, VH1-46, VH5-10-1 and Vkl-9 and VK3-11. mAbl8 humanized chains were designed using the germlines VH 1-46, VH5-10-1, VK1-9, Vk3-11 and Vk621. seven heavy chains (SEQ ID NOs: 40-45 and 89) and six light chains have been generated (SEQ ID NOs: 46-50 and 90).

[0393] Generation of Bispecific antibodies

[0394] The VH / VL sequences from mAbl7 and mAbl8 were designed in a Bispecific (BsAbs) BiXabs format (figure 1). The BsAb have four antigen binding sites: 2 distal Fabs from anti-HER2 Ab (External Fab) and 2 proximal Fabs from anti-iTCR Ab (internal Fab position). Three BsAbs, named BiXab-1, BiXab-2 or isotype, were designed as follows :

[0395] BiXab-1 : external Fab from trastuzumab (anti-HER2) and internal Fab from mAbl7 ; BiXab-2 : external Fab from trastuzumab (anti-HER2) and internal Fab from mAbl8 ; Isotype : external Fab from trastuzumab (anti-HER2) and an irrelevant internal Fab.

[0396] The external and internal Fab are separated by a linker, as described in the Table 1. The VH1- CH1 -Linker- VH2-CH1 were synthetized and cloned in pcDNA3.4 vector containing the Fc constant domain of SEQ ID NO: 88 (human IgGl LALA). Table 1: Sequence design of bispecific constructs

[0397] Expression, purification and quality control

[0398] Recombinant antibodies, monoclonals and Bixabs, were produced by transient transfection of ExpiCHO cells (Thermofisher) with the corresponding heavy and light chain encoding-plasmid using 1:1 molecular ratio for HC:LC of mAb and 1:1:1 molecular ratio for HC:LC1:LC2 for BsAb. A total of 25pg of DNA mixed with Expifectamine is used for 25mL transfection of ExpiCHO cells at 6 x 106cells. ExpiCHO feed and enhancer were added after 24h incubation in a 37° C incubator with a humidified atmosphere of 8% CO2 in air and under orbital shaking. Supernatants containing expressed recombinant antibodies were harvested between 8 to 10 days after transfection. Antibodies purified by affinity chromatography using protein A. Briefly, protein A Sepharose beads solution were incubated with the supernatant at least 3h before loading on a column. The antibodies were eluted in 0.1M glycine pH 3 and directly neutralized in 1:10 (v:v) Tris IM pH 8 buffer. Then size exclusion chromatography (SEC) was performed using AKTA. Antibody concentration was determined by Absorbance at 280 nM after filtration on 0.22pM and the quality of the antibody was assessed by SDS-PAGE in reduced and nonreduced condition and by analytical SEC.

[0399] Example 2: Screening and Binding against the recombinant anti-iTCR

[0400] Materials and methods Two human recombinant TCR were designed with the Voc7.2 chain comprising different CDR3a (SEQ ID NO: 68 named CDR3a-TCRl and SEQ ID NO: 69 named CDR3a-TCR2) and associated with either the V|313 (SEQ ID NO: 64 named human TRBV6-1-TRBJ2-2) or Vb2 (SEQ ID NO: 62 named human TRBV20-1). Both recombinant TCR present a His tag at the C- terminal part of the beta chain and a mouse IgG2a Fc tag (SEQ ID NO: 70 named mouse IgG2a Fc) at the end of the alpha chain. An “irrelevant” TCR using the same V(313 chain (SEQ ID NO: 64 named human TRBV6-1-TRBJ2-2) and the alpha chain Vocl2 (SEQ ID NO: 65) has been produced to evaluate the specificity for the Va7.2 chain. All the soluble TCR were produced by transient transfection of CHO cells and purified by affinity and size exclusion chromatography. Quality controls were performed to ensure the integrity of the protein. 0.05pg / wells of the recombinant TCR were coated in Maxisorp 96-well plates (overnight incubation at 4°C). After washes in PBS-0.05%Tween (PBS-T) the plates were saturated in PBS-2%BSA for 2h at room temperature (RT). After 3 washes in PBS-T, antibodies diluted in in PBS-0.5% BSA were added for Ih incubation at RT. After 3 washes in PBS-T, the secondary antibody HRP coupled anti-human IgG (H+L) (JIR # 109-036-170) was added at 1 / 5000 for Ih at RT. After 4 washes in PBS-T, the signal was revealed by TMB for 5min and stop solution (0.16M hydrochloric acid) before reading the optical density at a wavelength of 450 nm.

[0401] Screening of the anti-Va7.2 antibodies

[0402] First, the binding to the human TCR Voc7.2 / V|313 was assessed using the protein TRAV1-2- 2J33 / TRBV6-l-his and the specificity was assessed using an irrelevant TCR composed of the same beta chain and another alpha chain (Vocl2 / V|313) by direct ELISA. The 20 chimeric antibodies were evaluated and compared to the 3C10 clone (positive control) and an Isotype IgGl (negative control) at 100 nM (Figure 2).

[0403] Figure 2 shows that 8 antibodies bind only to the Voc7.2 / V|313 (FIG 2B) and not the Va 12 / V|313 (FIG 2A), as the 3C10 clone. Among the 8 specific antibodies against the Va7.2 chain, antibodies mAbl, mAb7, mAbl3, mAbl7 and mAbl8 showed significant absorbance signal > 5x threshold background.

[0404] Titration against the recombinant TCR

[0405] The 5 specific and high antibody binders identified in example 1 were titrated in serial dilution for their binding to the human recombinant Va7.2 TCR associated with either the VQ 13 or V|320 (Figure 3 A or 3B). GraphPad Prism software was used to plot the absorbance signal against the mAb concentration (nM). Resulting binding curves showed similar profiles against the 2 proteins Voc7.2TCR / V|313 (Figure 3A) and the Va7.2 TCR / V|32 (Figure 3B). We confirm all antibodies bind to the Va7.2 chain independently of the beta chain.

[0406] Curves were analyzed using the four-parameter logistic equation model for the calculation of EC50 (Table 2). The 5 antibodies showed different apparent affinity, lower, similar or higher than the 3C10 antibody. mAb 17 and mAb 18 show the lowest EC50 for both proteins, that is 3 to 4-fold lower than the 3C10. Those 2 antibodies are more potent than the original 3C10 clone to bind to the recombinant Va7.2 TCR, independently of the beta chain or the variability in the TCR CDR3a.

[0407] Table 2. EC50 values [nM] of anti-Va7.2 antibodies for their binding to the recombinant Va7.2 TCR measured by ELISA (Mean ofn=3 experiments).

[0408] Example 3: specific binding of the anti-Va7.2 antibodies to MAIT cells

[0409] MAIT cells expressing semi-invariant Va7 ,2 TCR can be identified by CD8+CD161Hi markers among T-cell population.

[0410] MAIT cells are the only cell type expressing exclusively the Va7.2 chain as shown using the MR1 tetramer and the Va7.2 co-staining (Figure 4). MAIT cells can be easily found in significant proportion in the CD8+ compartment among the cells expressing high CD161 markers. Indeed, the CD8+ T-cells recognizing the MR1-50PRU tetramer are all CD161Hi and Va7.2 positives. Thus, Va7.2+ primary cells are enriched using CD8+ T cells fraction and the CD 161 marker that identifies the MAIT cell population (Figure 4).

[0411] Material and method Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from enriched lymphocyte preparation of human healthy donor (Buffy coat), a side product of the blood banks collection. About 250 million cells were then enriched for CD8+ using a positive selection with CD8+ magnetic beads (Biolegend). This positive fraction was used to determine the binding of anti-Va7.2 antibodies to primary Va7.2+ MAIT cells (CD161HiCD8+). About 250 million of the PBMCs from the same donor were also depleted in Va7.2+cells using negative selection. Briefly, the 3C10-PE labelled antibody (4.5pL for 10M PBMCs) was incubated 20 min at 4°C. Then anti-PE microbeads (Miltenyi, 20 pLfor 10 million cells incubated 15min at 4°C) were used to recover the depleted Va7.2-PBMCs in the negative fraction. Those cells depleted of PBMC were used to assess the specificity to Va7.2+ cells. The anti-Va7.2 antibody binding by flow cytometry was performed using 0.1M immune cells (CD8+ or Va7.2-PBMCs). Briefly, cells were washed in PBS and incubated with Fixable viability dye (Live dead cell yellow, 1 / 1000, Thermofisher) and the human Fc block reagent (1 / 50 Biolegend) in PBS, 0.5mM EDTA, 0.5% BSA for 15min at room temperature. Cells were washed in PBS and stained with 100 nM of antibody or a range of concentration from 200 to 0.001 nM for 30 min at 4°C. The 3C10 clone and isotype human IgGl were used respectively as positive and negative control. After 2 washes, the cells were stained with a secondary antihuman antibody conjugated with Phycoerythrin (Jackson Immunoresearch) and a mix of anti- CD8-Pacific blue and anti-CD161-APC antibodies for CD8+ enriched cells or anti-CD3- AF700, anti-Va7.2-PE, anti-CD8-Pacific bleu, anti-CD4-PeCy7 antibodies for depleted PBMC (Biolegend). Cells were stained with the mix panel for 30 min at 4°C. After washing, cells were fixed in PBS+ 0.5% PFA and analyzed by flow cytometry using the Novocyte (Agilent) and the NovoExpress software.

[0412] Results

[0413] Specificity of the anti-Va7.2 antibodies to Va7.2+ cells using enriched CD8+ and Va7.2- PBMCs

[0414] The two anti-Va7.2 mAbl7 and mAbl8 produced in Example 1 and selected in Example 2 were tested for their binding to MAIT cells (Fig 5A) defined as CD161HiCD8+ cells (Figure 4) and their specificity for the Va7.2 TCR using Va7.2- PBMCs (Fig 5B) by flow cytometry. For this, the CD8+ enriched T-cells population and the Va7.2- PBMCs population of the same four healthy donors were used (donor 1 to 4). After isolation of the different population, immune cells were phenotyped to assess the frequency of Va7.2+ cells in the CD8+ enriched population and the residual Va7.2+ cells in the depleted PBMCs (Figure 5 A and 5B). The MAIT frequency is variable between donors, ranging from 4 to 38% among CD8+ cells in the 4 donors tested. We confirmed that after depletion of Va7.2+ cells, almost no cells were detected in the PBMCs with only 0.07-0.36% of residual Va7.2+ cells in the T-cells population.

[0415] The mAbl7 and mAbl8 binding was assessed by the signal of the secondary anti-human IgG coupled to PE fluorochrome. In the CD8+ enriched population, the PE fluorescence was assessed among the CD161Hi CD8+ T-cells, defined as the MAIT cells (Figure 5A) and in the Va7.2-PBMCs the signal was assessed among CD3+ T-cells (Figure 5B). Overlay of the mAb (Black) and the Isotype (Grey) are shown to visualize the background / specific signal.

[0416] Histograms showed no differences of binding at saturating concentration and specificity for the Va7.2+ TCR for mAbl7, mAbl8 or 3C10. In figure 5A, the peak shifts to the right compared to the isotype by 2-log in all donors, regardless of the MAIT frequency. The Va7.2- PBMCs from the same donor used to assess the potential background binding to other TCR showed similar histograms for all antibodies., 3C10, mAbl7, mAbl8 and the isotype (Fig. 5B). We concluded that no binding was detected and mAb 17 and mAb 18 are specific to the Va7.2 TCR receptor of MAIT cells.

[0417] Titration of the antibodies for their binding to MAIT cells

[0418] The two mAb were titrated for their binding to Va7.2+ primary cells (CD161HiCD8+) by flow cytometry. The mean fluorescence intensity value (MFI), extracted among CD161HiCD8+ cell population, has been reported for each mAb (Figure 6). Results show very similar binding profile for mAb 17 and mAb 18 for all donors with MFI plateau reached at about 10 nM. In comparison, the 3C10 showed a similar maximal MFI value for donor 3, donor 5 and donor 6 but reached the plateau at higher concentration (about 100 nM), and lower maximum MFI for donor 4. The calculated EC50 from the 4 donors performed in 2 independent experiments are reported in table 3. The EC50 is about 5.9 nM for the 3C10 compared to 0.77 and 1.03 nM for the mAb 17 and mAb 18 respectively. The values obtained are very homogeneous between donors and were not influenced by the initial MAIT frequencies. Those data are consistent with the data obtained by ELISA in Figure 2.

[0419] In conclusion, we confirm mAb 17 and mAb 18 are specific binders to MAIT cells, with similar specificity than 3C10 and higher potency. Table 3. EC50 values [nM] of anti-Va7.2 antibodies for their binding surface to Va7.2+ cells from 4 donors measured by flow cytometry.

[0420] Example 4: competition with the 3C10 antibody for their binding to Va7.2 mAbl7 and mAbl8 were tested for their ability to compete with the 3C10 antibody for their binding to Va7.2 TCR. This is assessed by the 3C10 binding signal in presence of the other mAb at different concentration by ELISA.

[0421] Material and method

[0422] For ELISA competition, 0.05pg / wells of the recombinant TCR Voc7.2 / V|313 was coated in Maxisorp 96-well plates (ON incubation at 4°C). After washes in PBS-0.05%Tween (PBS-T) the plates were saturated in PBS-2%BSA for 2h at RT. After 3 washes in PBS-T, the mAb 17, mAbl8, 3C10 or isotype antibodies at different concentration (starting from 150 nM, 5-fold dilution in PBS-0.5%BSA) were mixed with the 3C10-Biotin at 1.5 nM and added for Ih incubation at RT. After 3 washes in PBS-T, the secondary streptavidin coupled with HRP (Biolegend # 405210) was added at 1 / 3000 for Ih at RT. After 4 washes in PBS-T, the signal was revealed by TMB for 5min and stop solution (IM hydrochloric acid) before reading the optical density at a wavelength of 450 nm.

[0423] Results

[0424] Results are displayed in figure 7. Both mAbl7 and mAbl8 appear to compete with 3C10. Steric hindrance and / or higher affinity could explain this difference compared to the 3C10 mAb. Example 5: Cross-reactivity of the anti-Va7.2 antibodies to Non-Human Primate MAIT TCR

[0425] Cross-reactivity to the NHP Va7.2 TCR was determined by ELISA using the recombinant protein. Monovalent NHP recombinant TCR was produced by transient transfection of CHO cells with pcDNA3.1 vector carrying the Va7.2 chain that is identical for the cynomolgus and Rhesus monkey species (SEQ ID NO: 66) and the beta chain (SEQ ID NO: 67) and purified as described in the example 2.

[0426] Figure 8 shows the anti-Va7.2 antibodies binding to the NHP Va7.2 TCR evaluated by ELISA. As described in the literature, the 3C10 clone is a very low cross-reactive antibody. We observed a loss of binding of about 100-fold compared to the human Va7.2 TCR with no saturation reached at high concentration (FIG 8). On the contrary, Mabl7 and Mabl8 cross-react with the NHP TCR with a nanomolar apparent affinity. The calculated EC50 for mAbl7 and mAbl8 are 0.9 and 2 nM respectively (Table 4).

[0427] Table 4. EC50 value [nM] of cynomolgus Va7.2 TCR as measured, in 3 independent ELISA assay

[0428] EC50 (nM) mAbl7 mAbl8 3C10

[0429] Expl 0,445 2,212No

[0430] Exp2 0,198 0,897 saturation at

[0431] Exp3 0,777 1 ,927 350 nM

[0432] MEAN 0,474 1 ,679 NA

[0433] ET 0,291 0,692

[0434] To further illustrate the binding properties of mAbl8 antibody compared to the 3C10 antibody, SPR analysis has been performed in Single Cycle Kinetic using white fox instrument. Briefly, Streptavidin biosensors (ref #30.0007, Fox Biosystems) has been used and loaded with Biotinylated VHH anti human Fc (Proteintech, ref shurbGB-1) at 50 nM. Then, antibody 3C10 or mAbl8 was loaded at 50 nM for 180 sec. After 300 sec in the running buffer PBS 0.1% BSA (baseline), increasing concentrations of antigens human TCR or monkey TCR (6.25 nM, 12.5 nM, 25 nM, and 50 nM, diluted in BBS with 0.1% BSA) were successively incubated, with an association time of 180 seconds for each, followed by dissociation monitored for 600 seconds in PBS with 0.1% BSA. A 1 : 1 fit binding model was used to calculate kon, koff, and KD, reported in the table 5. mAbl8 antibody exhibits a higher affinity to human TCR (KD = 1.8 nM) compared to the3C10 antibody (KD = 7.8 nM) which is primarily due to a faster dissociation rate (koff) of the 3C10 antibody. This indicates that mAbl8 antibody forms a more stable complex with the MAIT TCR, whereas the3C10 antibody dissociates more rapidly. mAbl8 also showed cross-reactivity with the monkey TCR, with a significant KD of 20 nM. In contrast, the 3C10 antibody was completely unable to bind to the monkey TCR, with no association detected. This demonstrates a clear distinction between the mAbl8 antibody and the 3C10 antibody, with respect to both binding affinity and cross -reactivity.

[0435] Table 5. Kinetics and. affinity parameter of antibodies for human and monkey TCR evaluated by SPR

[0436] Example 6: simultaneous binding of the anti-Va7.2 / anti-HER2 antibodies to HER2 and Va7.2 in vitro

[0437] Bispecific antibodies have been designed in the BiXAb format as shown, in figure 1. The BiXAb-1 (HER2 x Voc7.2 Mabl7), BiXAb-2 (HER2 x Voc7.2 Mabl8) and BiXAb Isotype (HER2 x irrelevant antigen) has been designed as described in Table 1 and produced as described in Example 1.

[0438] The ability of the anti-HER2 x anti- Va7.2-based bispecific antibodies to bind both receptors was assessed in vitro by a dual ELISA. In this assay, the recombinant Voc7.2 / V|32 TCR (SEQ ID NO: 61 and SEQ ID NO: 62) was coated on ELISA plate at 0.05 pg. After blocking in PBS BSA 2% a range of BiXAbs concentration from 100 to 0.001 nM were incubated for Ih at RT. After 3 Washes in PBS-T, the recombinant HER2-His protein (Sino biological) was added at 1 pg / mL and incubated for Ih. After washes, the anti-His-HRP (Abeam) was used at 1 / 10000 dilution and incubated for Ih. TMB and stop solution (Thermofisher) were added after washes to read absorbance at 450 nm on spectrophotometer.

[0439] Figure 9 displays similar binding profile of both BiXAb-1 and BiXAb-2. They simultaneously bind to HER2 and the Va7.2 TCR. Moreover, they show similar apparent affinity than monoclonal mAbl7 and mAbl8 against the Va7.2 TCR alone in direct ELISA (figure 3).

[0440] Example 7: Redirected MAIT cell by the BiX b MAIT engager induce HER2+ cancer cells killing

[0441] The anti-Va7.2 x anti-HER2 BiXAbs were analyzed for their ability to induce lysis of HER2- expressing tumor cell.

[0442] Material and Method

[0443] To evaluate effector cell-mediated killing, SKBR3 Halo tag-Hibit cells (Promega, CS3057A32) was used according to the recommendation of the supplier. Briefly, target cell line was coculture with primary CD8+ enriched cells as effector cells at E / T ratio 5:1 with a range of BiXAb concentration from 0.001 to 3 nM. Controls without antibody and with Digitonin were included to determine the basal and maximum Hibit release, respectively. After 18 h incubation at 37°C, Nano-Glo(R) HiBiT Extracellular Detection System (Promega) was added directly to the wells for 10 min before luminescence measurement using the Infinite 200 pro reader (Tecan). Percentage of lysis was calculated as follows: % lysis = (experimental RLU - basal RLU) / (maximal RLU - basal RLU). Using GraphPad Prism software, the percentage of cytotoxicity relative to the maximal lysis control was plotted against BiXAb concentration (Fig 10).

[0444] Results

[0445] Figure 10 displays an example of cytotoxicity for the 2 anti-HER2 x anti-Va7.2 based bispecific antibodies compared to the isotype control. Dose response curves were analyzed with the four- parameter fitting model for evaluation of the sigmoidal curve. Both antibodies shown high potency to redirect MAIT cells and induce tumor cell killing up to 80% at 0.3nM. Example 8: Potency of redirecting non activated T cell against Ovarian cancer cell line OVCAR3

[0446] Material and Method

[0447] The ability of the anti-Va7.2 x anti-HER2-based bispecific antibodies (described in example 1 and 6 to redirect MAIT cells) was assessed for MAIT activation, MAIT degranulation and MAIT proliferation using flow cytometry readout and the release of INF-y by ELISA in coculture with HER2 expressing cell line OVCAR3. In this assay, OVCAR3 were cocultured with human enriched CD8+ T cells as effector cells (prepared as described in example 3) and an increasing concentration of bispecific antibodies or isotype (produced as described in example 1) from 0.001 nM to 3nM. A coculture of 20h at an effector target ratio of 2: 1 was used to look at activation markers CD69, CD 137 and INFy released ELISA kit (Biolegend# 430101). Degranulation of MAIT cells was assessed in a 5h-coculture assay with E / T 1:2 using the CD107a marker and GolgiSTOP (BD # 554724) was added 4h before the end of coculture. To determine the proliferation of MAIT and non MAIT cells, enriched CD8+ T cells were labelled with 2pM CFSE (Invitrogen # C34570) prior to coculture with target cells and the bispecific antibodies at an E / T of 1 : 1 for 6 days. Degranulation, activation and proliferation of MAIT cells was assessed at the end of the coculture by flow cytometry via the detection of CD107a, CD69 and CD137 markers among CD161HICD8+ or CFSE- among CD161HiCD8+ respectively. Briefly, surface cell staining was performed using a mix of anti-CD8 (Biolegend) and anti- CD161 (Miltenyi) with anti-CD69, anti-CD137 or anti-CD107a for staining 30min incubation at 4°C before flow cytometry measurement.

[0448] Results

[0449] Figures 11-14 show induction of dose-dependent MAIT cells degranulation (FIG 11), MAIT cells activation (FIG 12), secretion of INF-y (FIG 13) and proliferation of MAIT cells (FIG 14) specifically on MAIT cells and in the presence of OVCAR3. No effect was observed on non- MAIT conventional CD8+ T cells called CD8+ conv (CD161neg / low) in the presence of the BiXAbs and the cancer cells. No effect was observed on MAIT cells with the Isotype control (anti-HER2 x irrelevant antigen) showing the specificity of these molecules. 9: Mutational Yeast Surface

[0450] This example illustrates how the Epitope of the mAh 17 and mAh 18 antibodies have been identified. Specifically, this example shows epitope mapping by Deep Mutational Scanning (DMS) technology based on the use of DNA library of human Va7.2 TCR harboring a single mutation at each position, substituted by each possible amino acid, and screened by Yeast Surface Display (YSD). This library of TCR variants has been used for mAbl7 and mAbl8 binding by flow cytometry.

[0451] Briefly, yeasts expressing single mutant of the TCR were used for antibody binding. 10 million yeast cells were washed and incubated for 2h with the mAbl7 at 1 nM and mAbl8 at l.lnM. After 15 min incubation on ice, anti-V5 APC (TCR expression) or anti-human Fc PE (antibody binding) were added to reveal the TCR expression or the anti-Va7.2 mAb binding respectively. Cells were washed and resuspended in PBS 0.1% BSA to be analyzed by flow cytometry. The cells of interest that expressed the TCR but lost the antibody binding were sorted by FACS. Plasmid DNA of each yeast population was extracted and prepared as described in Medina- Cucurella and Whitehead (Medina-Cucurella A V., et al. 2018), for deep sequencing with Illumina MiSeq device. After sequencing data analysis using dedicated bioinformatic scripts, the enrichment ratios for each single mutation were calculated and obtained. Then, for each amino acid position, a score from 0 to 19 has been settled as the sum of the number of forbidden substitutions (19 being the maximum possible). The epitope is defined by all the amino acids of the TCR that are both exposed to solvents and with a score above 11.

[0452] The predicted 3D structure of the MAIT TCR and the epitope are shown in figure 15 using AlphaFold software. The amino acids that are part of the epitope are highlighted in bold and shown in black. Amino acids E51, D52, G53, E54, F63, S65, Y72, S62, E55, E56 and R66 are critical residues involved in the mAbl7 binding. Amino acids E51, D52, K68, G53, E54, F63, S62, E55 and R66 are critical residues involved in the mAb 18 binding. The key epitope position for mAb 17 are the amino acids G53, E55 and Y72, while for mAb 18 there are D52, G53, E55, F63, R66, that are critical residues involved in their respective binding.

[0453] 10. Binding capacity of mAbl8 variants to Va7.2 TCR of MAIT cells Two humanized mAbl8 variants, designated as “variant 1” and “variant 2” were produced using a human IgGl scaffold and tested to confirm their biological properties.

[0454] The variable heavy chain of mAbl8 variant 1 is shown in SEQ ID NO: 43, with H-CDR1, H- CDR2 and H-CDR3 consisting of SEQ ID NOs: 4, 5 and 6, respectively. The variable light chain of mAbl8 variant 1 is shown in SEQ ID NO: 49, with L-CDR1, L-CDR2 and L-CDR3 consisting of SEQ ID NOs: 87, 19 and 20, respectively.

[0455] The variable heavy chain of mAbl8 variant 2 is shown in SEQ ID NO: 89, with H-CDR1, H- CDR2 and H-CDR3 consisting of SEQ ID NOs: 4, 91 and 92, respectively. The variable light chain of mAbl8 variant 2 is shown in SEQ ID NO: 90, with L-CDR1, L-CDR2 and L-CDR3 consisting of SEQ ID NOs: 87, 19 and 93, respectively.

[0456] This example illustrates mAbl8 variant 1 and variant 2 binding to the TCR of MAIT cells by ELISA or flow cytometry. As described in example 2, the titration of both variants was performed for their binding to the human TCR Voc7.2 / V|313 by ELISA. mAbl8 variants exhibit similar binding profile (Figure 16A), with comparable EC50 values (0.032 nM and 0.034 nM for mAbl8 variants 1 and 2 respectively). mAbl8 variants were also assessed in titration for binding to primary MAIT cells by flow cytometry, as described in example 3. Figure 16B displays the titration curve using a range of antibody concentration (100-0. OOlnM) to assess apparent avidity of both variants. In concordance with previous data, both variants exhibit similar binding profile to MAIT cells with increase signal in dose dependent manner. Similar EC50 values were measured (0.8 nM and 1 nM for variant 1 and variant 2 respectively). variants to induce MAIT cells activation and

[0457] This example illustrates the biological properties of mAbl8 variants in activating MAIT cells via presentation by THP-1 cells, looking at degranulation and upregulation of activation marker CD69 and CD 137 on MAIT cells. Briefly, THP-1 cells were seeded at 10A5 cells / well in V- bottom 96-well plates and incubated with a range of antibodies (4nM-0.04nM final) for 30min at RT. After washes, THP-l-mAb were incubated with 0.2xl0A6 human CD8+ effector cells in U-bottom 96-well plates to initiate the coculture at 37°C. To monitor degranulation, anti- CD107a-AF488 antibody was added to the THP-1 / CD8+ cell suspension at 1 pL / well for Ih at 37°C, 5% CO2, followed by addition of GolgiStop™ (monensin, 1 / 1500) and incubation for an additional 4 h. After incubation, cells were harvested, stained with viability dye, CD8, and CD 161, washed, fixed, and analyzed by flow cytometry. MAIT cell degranulation was quantified as CD107a expression. To measure upregulation of activation markers, cocultures were incubated for 18-24 h at 37 °C, 5% CO2. Cells were then harvested, washed, and stained with viability dye together with CD8-BV421, CD161-APC, CD69-FITC, and 4-lBB-PE-Cy7. After incubation for 30 min at 4 °C, cells were washed, resuspended, and acquired by flow cytometry. MAIT cell activation was quantified based on CD69 and CD137 expression.

[0458] Figure 17 displays the degranulation of MAIT cells induced by mAbl8 variant 1 or variant 2, or by the 3C10 antibody, presented by THP-1 cells. These data show that both mAbl8 variants induce similar MAIT cells degranulation, whereas the 3C10 antibody exhibits a reduced capacity to induce MAIT cells degranulation, as shown by lower expression levels of CD 107a. This represents 45% of degranulation of MAIT cells induced by mAbl8 variants vs 17% induced by 3C10 antibody (data not shown).

[0459] Figure 18 shows the upregulation of CD 137 (Figure 18 A) and CD69 (Figure 18B) induced by mAbl8 variant 1 or variant 2, or the 3C10 antibody. The results show that both mAbl8 variants induce high levels of CD 137 and CD69 on MAIT cells, in a dose dependent manner. In contrast, the 3C10 antibody induces a significantly lower activation, with levels of CD 137 and CD69 reduced by two- to three-fold.

[0460] References

[0461] Chari, R. V., Martell, B. A., Gross, J. L., Cook, S. B., Shah, S. A., Blattler, W. A., McKenzie, S. J., & Goldmacher, V. S. (1992). Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer research, 52(1), 127-131.

[0462] Dusseaux M, Martin E, Serriari N, Peguillet I, Premel V, Louis D, Milder M, Le Bourhis L, Soudais C, Treiner E, Lantz O. Human MAIT cells are xenobiotic -resistant, tissue-targeted, CD161hi IL-17-secreting T cells. Blood. 2011 Jan 27; 117(4): 1250-9. doi: 10.1182 / blood-2010- 08-303339. Epub 2010 Nov 17. PMID: 21084709. Franciszkiewicz K, Salou M, Legoux F, Zhou Q, Cui Y, Bessoles S, Lantz O. MHC class I- related molecule, MR1, and mucosal-associated invariant T cells.Immunol Rev. 2016 Jul;272(l): 120-38.

[0463] Koristka S, Cartellieri M, Theil A, Feldmann A, Arndt C, Stamova S, Michalk I, Topfer K, Temme A, Kretschmer K et al.: Retargeting of human regulatory T cells by single-chain bispecific antibodies. J Immunol 2012, 188:1551-1558

[0464] Le Bourhis et al, PLoS Pathogens, 2013, Volume 9, Issue 10, el003681

[0465] Reantragoon R, Corbett AJ, Sakala IG, Gherardin NA, Furness JB, Chen Z, Eckle SB, Uldrich AP, Birkinshaw RW, Patel O, Kostenko L, Meehan B, Kedzierska K, Liu L, Fairlie DP, Hansen TH, Godfrey DI, Rossjohn J, McCluskey J, Kjer-Nielsen L. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J Exp Med. 2013 Oct 21;210(ll):2305-20. doi: 10.1084 / jem.20130958. Epub 2013 Oct 7. PMID: 24101382; PMCID: PMC3804952.

[0466] Salou M, Franciszkiewicz K and Lantz O: MAIT cells in infectious diseases. Curr Opinion Immunol, 2017;48:7-14.

[0467] Salou et al, J Exp Med. 2019 ;216(1): 133-151

[0468] Tilloy F, Treiner E, Park SH, Garcia C, Lemonnier F, de la Salle H, Bendelac A, Bonneville M, Lantz O. An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha / beta T cell subpopulation in mammals. J Exp Med. 1999 Jun 21 ; 189(12): 1907-21. doi: 10.1084 / jem. l89.12.1907. PMID: 10377186; PMCID: PMC2192962. van de Donk NWCJ, Zweegman S. T-cell-engaging bispecific antibodies in cancer. Lancet. 2023 Jul 8;402(10396): 142-158. doi: 10.1016 / 80140-6736(23)00521-4. Epub 2023 Jun 1. PMID: 37271153.

[0469] Wahl, R. L., Parker, C. W., & Philpott, G. W. (1983). Improved radioimaging and tumor localization with monoclonal F(ab')2. Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 24(4), 316-325.

Claims

74Claims1. An antigen binding fragment that specifically binds to a polypeptide comprising Va7.2, wherein said antigen binding fragment comprises a heavy chain comprising the following heavy chain complementarity-determining regions (H-CDRs):(i)H-CDR1 that shows sequence SEQ ID NO: 1 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 1 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 1;H-CDR2 that shows sequence SEQ ID NO: 2 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 2 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 2; andH-CDR3, that shows SEQ ID NO: 3 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 3 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 3; or(ii)H-CDR1 that shows sequence SEQ ID NO: 4 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 4, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 4;H-CDR2 that shows sequence SEQ ID NO: 5 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 5, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 5; andH-CDR3 that shows sequence SEQ ID NO: 6 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 6, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 6.

2. The antigen binding fragment of claim 1, wherein the heavy chain further comprises :- in (i), framework regions that show at least 80% identity with the framework regions set forth in SEQ ID NO: 7 to 9 and SEQ ID NO: 12; or,- in (ii), framework regions that show at least 80% identity with the framework regions set forth in SEQ ID NO: 8 and SEQ ID NO: 10 to 12 ; respectively.

753. The antigen binding fragment of claim 1 or 2, wherein the antigen binding fragment comprises : in (i), a heavy chain variable region sequence as set forth in SEQ ID NO: 13, or having at least 80%, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 13 ; or in (ii), a heavy chain variable region sequence as set forth in SEQ ID NO: 14, or having at least 80%, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 14.

4. The antigen binding fragment of any of claims 1 to 3, which further comprises a light chain comprising the following light chain complementarity-determining regions (L-CDRs) : in i),L-CDR1 that shows sequence SEQ ID NO: 15 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 15, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 15;L-CDR2 that shows sequence SEQ ID NO: 16 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 16, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 16; andL-CDR3, that shows SEQ ID NO: 17 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 17 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 17; or, in ii)L-CDR1 that shows sequence SEQ ID NO: 18 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 18, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 18;L-CDR2 that shows sequence SEQ ID NO: 19 or a variant thereof, said variant having at least 80% sequence identity with SEQ ID NO: 19, and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 19; andL-CDR3, that shows SEQ ID NO: 20 or a variant thereof, said variant having at least 80% sequence identity with respect to SEQ ID NO: 20 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NO: 20, respectively ; optionally wherein the light chain further comprises76 in (i), framework regions that show at least 80% identity with the framework regions set forth in SEQ ID NO: 21 to 24; or, in (ii), framework regions that show at least 80% identity with the framework regions set forth in SEQ ID NO: 25, 26, 23 andto 28 respectively.

5. The antigen binding fragment of any of claims 1 to 4, wherein the antigen binding fragment comprises : in i), a light chain variable region sequence as set forth in SEQ ID NO: 29, or having at least 80%, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 29; or, in ii), a light chain variable region sequence as set forth in SEQ ID NO: 30, or having at least 80%, preferably at least 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 30.

6. The antigen binding fragment of claim 1, which is a humanized antigen binding fragment, preferably wherein said humanized antigen binding fragment comprises :- in i) H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, SEQ ID NO: 78, SEQ IDNO: 79 or SEQ ID NO: 80, and H-CDR3 of SEQ ID NO: 3, or variants thereof having at least 80% identity to SEQ ID NOs: 1-3 and 78-80 ; and / or L-CDR1 of SEQ ID NO: 15 or SEQ ID NO: 85, L-CDR2 of SEQ ID NO: 16 or SEQ ID NO: 86, and L-CDR3 of SEQ ID NO: 17, or variants thereof having at least 80% identity to SEQ ID NOs: 15-17 and 85-86 ; or- in ii), H-CDR1 of SEQ ID NO: 4 or SEQ ID NO: 81, H-CDR2 of SEQ ID NO: 5, SEQID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 91, and H-CDR3 of SEQ ID NO: 6 or SEQ ID NO: 92, or variants thereof having at least 80% identity to SEQ ID NOs: 4-6, 81-84 and 91-92 ; and / or L-CDR1 of SEQ ID NO: 18 or SEQ ID NO: 87, L- CDR2 of SEQ ID NO: 19 and L-CDR3 or SEQ ID NO: 20 or SEQ ID NO: 93; or variants thereof having at least 80% identity to SEQ ID NOs: 18-20, 87 and 93; respectively ; preferably, wherein said humanized antigen binding fragment comprises :- in i), a variable heavy chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 31, 32, 33, 34 and 35, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 31-35 ; and / or a variable light chain comprising77 or consisting of a sequence selected in the group consisting of SEQ ID NOs: 36, 37, 38 and 39, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 36- 39 ; or- in ii), a variable heavy chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 40, 41, 42, 43, 44, 45 and 89, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 40-45 and 89; and / or a variable light chain comprising or consisting of a sequence selected in the group consisting of SEQ ID NOs: 46, 47, 48, 49,50 and 90, or variants thereof having at least 80% sequence identity to SEQ ID NOs: 46-50 and 90 ; respectively.

7. The antigen binding fragment of claim 6, which is a humanized antigen binding fragment comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 5 and H-CDR3 of SEQ ID NO: 6, or variants thereof having at least 80% sequence identity with SEQ ID NOs: 4-6 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NOs: 4-6 ; and / or comprising L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 20, or variants thereof having at least 80% sequence identity with SEQ ID NOs: 87, 19 and 20 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NOs: 87, 19 and 20, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 43, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 43 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 49, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 49.

8. The antigen binding fragment of claim 6, which is a humanized antigen binding fragment comprising H-CDR1 of SEQ ID NO: 4, H-CDR2 of SEQ ID NO: 91 and H-CDR3 of SEQ ID NO: 92, or variants thereof having at least 80% sequence identity with SEQ ID NOs: 4, 91 and 92 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NOs: 4, 91 and 92 ; and / or comprising L-CDR1 of SEQ ID NO: 87, L-CDR2 of SEQ ID NO: 19 and L-CDR3 of SEQ ID NO: 93, or variants thereof having at least 80% sequence identity with SEQ ID NOs: 87, 19 and 93 and / or having a substitution of one, two or three amino acids with respect to SEQ ID NOs: 87, 19 and 93, wherein, preferably, said humanized antigen binding fragment comprises : a variable heavy chain comprising or consisting of SEQ ID NO: 89, or a variant thereof having at least 80%78 sequence identity to SEQ ID NO: 89 ; and / or a variable light chain comprising or consisting of SEQ ID NO: 90, or a variant thereof having at least 80% sequence identity to SEQ ID NO: 90.

9. A protein construct which comprises the antigen binding fragment as defined in any one of claims 1 to 8, optionally linked to at least another polypeptide; optionally wherein the protein construct is a monoclonal antibody, a multivalent antibody or a multispecific antibody; preferably wherein the protein construct is an antibody that comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgGl, IgG2, IgG3 or IgG4 heavy chain constant domain (CHI).

10. The protein construct of claim 9, which comprises :- two identical antigen-binding arms each comprising, or consisting of an antigen-binding fragment as defined in any one of claims 1 to 8; and- an Fc region.

11. A multispecific molecule, preferably a bispecific molecule, that comprises the antigen binding fragment as defined in any of claims 1 to 8, or the protein construct of claims 9 or 10, wherein the molecule also comprises at least one domain that binds another antigen, preferably a tumor associated antigen (TAA), more preferably a TAA selected from the group consisting of CD19, EGFR, and HER2.

12. The multispecific molecule of claim 11, which comprises at least one multispecific antigenbinding fragment comprising at least two Fab fragments with different CHI and CL domains, wherein said Fab fragments are tandemly arranged in any order, the C-terminal end of the CHI domain of a first Fab fragment being linked to the N-terminal end of the VH domain of the following Fab fragment through a polypeptide linker, wherein at least one Fab fragment comprises the antigen-binding fragment as described in claims 1 to 8, and at least another Fab fragment binds another antigen, preferably a TAA, more preferably a TAA selected from the group consisting of CD 19, EGFR, and HER2.

13. An isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding the antigen-binding fragment, the protein construct or the multispecific molecule as defined in any one of claims 1 to 12.

14. A vector comprising the isolated nucleic acid molecule or the group of isolated nucleic acid molecules of claim 13.

15. A host cell comprising the isolated nucleic acid molecule or the group of isolated nucleic acid molecules of claim 13, or the vector according to claim 14.

16. A method for producing the antigen-binding fragment, protein construct or multispecific molecule of any one of claims 1 to 12, comprising a step of culturing a host cell according to claim 15 and optionally a step of isolating the antigen-binding fragment, protein construct or multispecific molecule.

17. The multispecific molecule as defined in any one of claims 11 to 12, for use in treating a cancer.

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