TW202200617A - Antibodies having specificity for btnl8 and uses thereof - Google Patents

Abstract

The present invention relates to antagonist antibodies having specificity for BTNL8 and uses thereof, in particular for the treatment of inflammatory diseases.

Description

Antibodies specific for BTNL8 and uses thereof

The present invention relates to anti-BTNL8 antibodies that bind to BTNL8 and inhibit Vδ1 T cell cytotoxicity. Such antibodies are particularly useful in the treatment of inflammatory diseases.

White blood cells are cells involved in the immune system that defends the body against pathogens. In addition to conventional MHC class I-restricted CD8+ CTL and NK cells, other non-conventional T cells, especially γδ T cells, showed the same sensitivity and lytic capacity as NK and T cells.

Butteroproteins and butyrophilin-like proteins (BTN/BTNL) are a family of members of the immunoglobulin superfamily that influence immunity, such as T cell selection, and developmental processes, such as differentiation and cell fate determination (Blasquez et al., Frontiers). in Immunology 2018, 9, 1601).

Di Marco Barros et al. (Cell 2016, 167, 203-218) reported that BTNL3 and BTNL8, as expressed in human intestinal epithelial cells, combined induce a selective TCR-dependent response in human colonic Vγ4/Vδ1 cells.

Melandri et al. (Nat Immunol. 2018, 19(12): 1352-1365) proposed that the conserved mechanism by which Btnl or BTNL proteins regulate gamma delta intraepithelial lymphocytes (IELs) is by coupling to a supporting chain (Btnl1 Or BTNL8) "interacting chain" (Btnl6 or BTNL3) mediated.

WO2019/234136 further reports the direct interaction of BTNL3/BTNL8 heterodimers with certain recombinant γ4-containing TCRs, including γ4δ1 TCRs.

WO2019/053272 also reports compositions and methods suitable for the treatment of intestinal inflammation by modulating γδ T cells (and eg Vγ4+ cells).

WO2019/057933 discloses antibodies specific for BTN2 and their use in methods for the treatment of autoimmune and inflammatory disorders.

To date, there remains a need to identify treatments for inflammatory diseases, such as those caused by intestinal inflammation, including but not limited to inflammatory bowel disease, celiac disease, Crohn's disease, or ulcerative colitis . More generally, there is a need to identify new inhibitors and/or methods of suppressing immune responses in patients in need.

The present invention discloses that certain anti-BTNL8 antibodies can inhibit Vδ1 T cell activity, specifically Vδ1 T cell cytotoxicity. Therefore, such antibodies are particularly useful in the treatment of inflammatory diseases.

In a first aspect, the invention relates to an antibody specific for human BTNL8 (BTNL8), characterized in that the antibody has one or more of the following properties: i. It binds to human BTNL8/BTNL3 dimers as expressed in cell lines, such as HEK-293T cells, and/or ii. It does not bind to cell lines expressing BTNL3 but not BTNL8, such as the HEK-293T cell line expressing BTNL3.

In particular embodiments, this antibody specific for human BTNL8 (BTNL8) binds to human _BTNL8 -Fc with a KD below 10 pM, eg, as measured by a Luminex assay.

In a preferred embodiment, the anti-BTNL8 antibody of the present invention has at least one of the following properties: i. It inhibits the activation of Vδ1 TCR-bearing T cells (Vδ1 T cells), ii. It inhibits the lytic function of activated Vδ1 T cells, and/or iii. It inhibits the production of cytokines by activated Vδ1 T cells.

In particular embodiments, the anti-BTNL8 antibodies of the invention have one or more of the following properties: i. It inhibits activation of Vγ4Vδ1 TCR-bearing T cells, typically as determined by a Vγ4Vδ1 TCR reporter cell assay; and/or ii. It inhibits the lytic function of activated Vδ1 T cells, generally it inhibits the degranulation of Vδ1 T cells against HL-60 cells, as determined in an in vitro degranulation cell assay;

In particular embodiments, the anti-BTNL8 antibodies of the invention further advantageously cross-react with cynomolgus monkey BTNL8.

Examples of such anti-BTNL8 antibodies include the following reference murine antibodies: i. Reference murine antibody mAb X1 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 variable area; ii. Reference murine antibody mAb X2 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 variable area; iii. Reference murine antibody mAb X3 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:6 variable area; iv. Reference murine antibody mAb X4 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8 variable area; or v. Reference murine antibody mAb X5 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:10 variable area.

Part of the invention is also an anti-BTNL8 antibody that competes for binding to BTNL8 with at least one of the reference antibodies defined above.

In specific embodiments, the anti-BTNL8 antibodies of the invention i. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X1 comprising SEQ ID NOs: 11 to 16, respectively; ii. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 comprising mAb X2 of SEQ ID NOs: 17 to 22, respectively; iii. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X3 comprising SEQ ID NOs: 23 to 28, respectively; iv. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X4 comprising SEQ ID NOs: 29 to 34, respectively; or v. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X5 comprising SEQ ID NOs: 35 to 40, respectively.

The anti-BTNL8 antibody according to any one of the foregoing technical solutions, which is an antibody comprising the following i. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:1 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:2; ii. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:3 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:4; iii. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:5 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:6; iv. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:7 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:8; or v. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:9 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:10.

Typically, the anti-BTNL8 antibody of the present invention can be a human, chimeric or humanized antibody.

In another aspect, the present invention relates to nucleic acid molecules encoding the heavy and/or light chains of an anti-BTNL8 antibody as defined above.

The present invention further provides a host cell comprising any one of the nucleic acid molecules encoding the anti-BTNL8 antibodies mentioned above.

The present invention also relates to an anti-BTNL8 antibody as described herein for use as a medicament, eg, for the treatment of an inflammatory disorder (eg, intestinal inflammation) in a subject. Preferred examples of such inflammatory disorders include, but are not limited to, inflammatory bowel disease (IBD), in particular Crohn's disease, celiac disease or ulcerative colitis.

The present invention further relates to a pharmaceutical composition comprising the anti-BTNL8 antibody of the present invention and at least one pharmaceutically acceptable carrier.

Definitions As used herein, the term "BTNL8" has its ordinary meaning in the art and refers to a human BTNL8 polypeptide including BTNL8 having SEQ ID NO:81. SEQ ID NO: 81: Homo sapiens butyrophilin-like 8 (BTNL8) MALMLSLVLSLLKLGSGQWQVFGPDKPVQALVGEDAAFSCFLSPKTNAEAMEVRFFRGQFSSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAEGRISLRLENITVLDAGLYGCRISSQSYYQKAIWELQVSALGSVPLISITGYVDRDIQLLCQSSGWFPRPTAKWKGPQGQDLSTDSRTNRDMHGLFDVEISLTVQENAGSISCSMRHAHLSREVESRVQIGDTFFEPISWHLATKVLGILCCGLFFGIVGLKIFFSKFQWKIQAELDWRRKHGQAELRDARKHAVEVTLDPETAHPKLCVSDLKTVTHRKAPQEVPHSEKRFTRKSVVASQSFQAGKHYWEVDGGHNKRWRVGVCRDDVDRRKEYVTLSPDHGYWVLRLNGEHLYFTLNPRFISVFPRTPPTKIGVFLDYECGTISFFNINDQSLIYTLTCRFEGLLRPYIEYPSYNEQNGTPIVICPVTQESEKEASWQRASAIPETSNSESSSQATTPFLPRGEM

As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and will be used equivalently in the present invention.

More specifically, the term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that immunospecifically binds an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.

As used herein, the term "antibody" also includes bispecific or multispecific molecules. An antibody can be derivatized or linked to another functional molecule (e.g., another peptide or protein (e.g., another antibody or ligand to a receptor)) to generate a molecule that binds to at least two different binding sites or target molecules. bispecific molecules. Antibodies may indeed be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be derived from, as described herein, The term "bispecific molecule" is used to encompass. To form bispecific molecules, the antibodies of the invention can be functionally linked (eg, by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules (such as another antibody, antibody fragments, peptides or binding mimetics), resulting in bispecific molecules. Additionally, for embodiments where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the first and second epitopes of interest.

In native antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (lambda; l) and kappa (kappa; k). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. A light chain includes two domains: a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains: a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light (VL) and heavy (VH) chains determine binding recognition and specificity for antigen. The constant regions of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcRs) .

Fv fragments are the N-terminal part of Fab fragments of immunoglobulins and consist of the variable part of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinants. The antibody binding site consists of residues derived primarily from hypervariable or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable or framework regions (FRs) can participate in the antibody binding site or affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv regions of the native immunoglobulin binding site.

The light and heavy chains of immunoglobulins each have three CDRs designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically includes six CDRs comprising the set of CDRs from each of the heavy and light chain V regions. Framework regions (FR) refer to amino acid sequences inserted between CDRs. Accordingly, the variable regions of light and heavy chains typically comprise 4 framework regions and 3 CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used in this manual. The Kabat residue designation does not always correspond directly to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional elements than in the strict Kabat numbering corresponding to or inserted into the structural components (framework regions or complementarity determining regions (CDRs)) that shorten the basic variable domain structure. amino acid. The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence to a "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), residues 50 to 65 (H-CDR2) and residues 95 to 102 (H-CDR3). According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24 to 34 (L-CDR1), residues 50 to 56 (L-CDR2) and residues 89 to 97 (L-CDR3).

In particular embodiments, the antibodies provided herein are antibody fragments, and more particularly any protein comprising the antigen binding domain of the antibodies as disclosed herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies.

As used herein, the term "specific for BTNL8" refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as BTNL8. In other embodiments, it is intended to refer to an antibody that binds to the heterodimeric BTNL8/BTNL3 as expressed in a cell line (eg, the HEK293T cell line as described in the Examples). In some embodiments, it is intended to refer to antibodies that: (i) bind to human BTNL8/BTLN3 dimers as expressed in a cell line (eg, HEK293T cell line) and (ii) do not bind to cells expressing BTNL3 but not BTNL8 strains, generally as described in the Examples. In other embodiments, it binds to the _BTNL8 recombinant polypeptide with a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less. In some embodiments, it is intended to refer to an antibody that binds to the human BTNL8-Fc protein as assessed by Luminex-based affinity assessment. More specifically, the antibody binds to the human _BTNL8 -Fc protein with a KD of less than 100 pM, more preferably less than 10 pM, as assessed by Luminex-based assessment as described in the Examples.

In some embodiments, the antibody specific for BTNL8 further does not cross-react with BTNL3.

An antibody that "cross-reacts with an antigen other than _BTNL8 " is intended to mean an antibody that binds that antigen with a KD of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody that "does not cross-react with a particular antigen (eg, _BTNL3 )" is intended to mean an antibody that binds to that antigen with a _KD of 100 nM or greater, or a KD of 1 μM or greater, or a KD of 10 _μM or greater . In certain embodiments, such antibodies do not cross-react with antigen in standard binding assays, exhibiting substantially undetectable binding to these proteins. Cross-reactivity can be tested by affinity binding assays with recombinant antigens (eg, recombinant BTNL3-Fc) or with cells expressing the antigen (eg, cell lines expressing BTNL3).

As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (eg, an isolated antibody that specifically binds to BTNL8 is substantially free of specific binding to other than BTNL8 antibodies to other antigens). However, isolated antibodies that specifically bind to BTNL8 may be cross-reactive with other antigens, such as related BTNL8 molecules from other species, eg, cynomolgus monkey BTNL8. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of monoclonal composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The phrases "antibody that recognizes an antigen" and "antibody specific for the antigen" are used interchangeably herein with the term "antibody that specifically binds to the antigen".

The term "_{Kassoc" or "Ka" as used herein is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kdis " or} " _Kd " as used herein is intended to refer to a particular antibody- Dissociation rates of antigen interactions.

As used herein, the term " _KD " is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of _koff to _kon (ie, _koff / _kon ), and is expressed in molar concentration (M). The _KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and thus, the lower the _KD value (lower concentration) and thus the higher the affinity of the antibody. The _KD value of an antibody can be determined using methods well established in the art. _A preferred method for determining the K of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Coligan et al., eds. Current Protocols in Immunology, In Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), these references are incorporated by reference in their entirety. One method for determining the _K of an antibody is by using surface plasmon resonance or by using a biosensor system such as ^Biacore® (see also Rich RL, Day YS, Morton TA, for details on affinity assessment, Myszka DG. High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE®. Anal Biochem. 2001 Sep 15;296(2):197-207) or the Octet® system. The Octet® platform is based on bio-layer interferometry (BLI) technology. The principle of BLI technology is based on an optical interference pattern of white light reflected from two surfaces (an immobilized protein layer and an internal reference layer). Binding between ligands immobilized on the surface of the biosensor tip and analytes in solution results in an increase in optical thickness at the biosensor tip, which causes a shift in the interference pattern measured in nanometers . The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biolayer, and when this shift is measured over a period of time and its magnitude is plotted as a function of time, a classical association/dissociation curve is obtained. This interaction is measured in real time, allowing monitoring of binding specificity, association and dissociation rates, and concentration. (See Abdiche et al. 2008 and details in Results). Affinity measurements are typically performed at 25°C. Another method can be Luminex analysis as described in the Examples.

When binding to a specific antigen relative to non-specific binding to other unrelated molecules (in this case, the specific antigen is a BTNL8 polypeptide), it can be further enhanced by, for example, about 10:1, about 20:1, about 50:1 , an affinity/avidity ratio of about 100:1, 10.000:1 or greater exhibits specificity. As used herein, the term "affinity" means the strength with which an antibody binds to an epitope.

As used herein, the term "identity" refers to sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When one of the positions in the two compared sequences is occupied by the same base or the same amino acid residue, then the respective molecules are identical at that position. The percent identity between the two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. In general, comparisons are made when two sequences are aligned for maximum identity. Alignment can be performed by using, for example, any of the GCG (Genetics Computer Group, Manual of Programs for GCG Packages Version 7, Madison, Wisconsin) stacking program, or sequence comparison algorithms such as BLAST, FASTA or CLUSTALW to calculate consistency.

As used herein, functional variants of a reference antibody according to the present invention exhibit functional properties that are substantially equal to or better than the corresponding functional properties of the reference antibody (eg, any of mAbs X1 to X5). By being substantially equal, it is intended herein that the functional variant exhibits at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the corresponding functional property of the reference antibody.

Specific Embodiments of Anti- BTNL8 Antibodies of the Invention In one aspect, the antibody specific for BTNL8 according to the invention may be further characterized as having at least one of the following properties: - It inhibits Vδ1 TCR-bearing T Activation of cells (Vδ1 T cells), - which inhibits the lytic function of activated Vδ1 T cells, and/or - which inhibits the production of interferons by activated Vδ1 T cells.

As used herein, "inhibiting activation of Vδ1 TCR-bearing T cells" means using TCRαβ-free T cells transduced to express colon-derived Vγ4Vδ1 TCR and the reporter gene in the presence of cell lines expressing BTNL8 and BTNL3 Cell line derivatives, significantly reduced expression of the reporter gene under the control of the NF-AT promoter was observed in the Vγ4Vδ1 TCR reporter cell assay. "Inhibition of activation of Vδ1 TCR-bearing T cells" can also be determined by measuring the reduction of CD69 expression at the plasma membrane or by measuring the inhibition of down-regulation of TCRVδ1 expression at the plasma membrane. An example of this reporter cell assay is described in more detail in the Examples below.

In some embodiments, the antibodies of the invention inhibit activation of Vγ4Vδ1 TCR-bearing T cells (Vγ4Vδ1 T cells) to an extent that is substantially different from at least one of the following reference antibodies as described below: mAb X1, mAb X2 , mAb X3, mAb X4 and mAb X5.

As used herein, "inhibiting the lytic function of activated Vδ1 T cells" means that a significant reduction in the lytic function of activated Vδ1 T cells is observed when compared to control activated Vδ1 T cells (of the control isotype). . In particular embodiments, the anti-BTNL8 antibodies of the invention inhibit the lytic function of activated Vδ1 T cells to a degree that is substantially equal to or better than the degranulation of basal Vδ1 T cells as described below for the HL-60 cell line . In a more specific embodiment, the level of lytic function of activated Vδ1 T cells with the tested anti-BTNL8 antibody is reduced to at least 5%, more specifically at least 10% and preferably, compared to a control isotype antibody at least 15%.

Thus, the anti-BTNL8 antibodies of the invention having such advantageous properties as defined above can be used in anti-BTNL8 antibodies using cellular assays as described in the Examples and in particular for the determination of Vδ1 T cells against BTNL8 expressing cancers Cell lines, such as the HL-60 myeloid leukemia cell line, were screened for analysis of modulation of degranulation.

In some embodiments, the antibodies of the invention inhibit the lytic function of activated Vδ1 T cells to an extent that is substantially equal to or better than at least one of the following reference antibodies as described below: mAb X1, mAb X2, mAb X3, mAb X4 and mAb X5.

As used herein, "inhibiting the production of interferons by activated Vδ1 T cells" means that interleukin (usually TNFα) is observed as compared to the production of interferons from activated Vδ1 T cells in the presence of a control isotype antibody. and/or IFNγ) production was significantly reduced.

Thus, in preferred embodiments, the anti-BTNL8 antibody of the invention exhibits at least one or more of the following functional properties: i. It is specific for BTNL8, in particular binding to cell lines as expressed, for example via human BTNL8 in HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8 (as described in the Examples), more specifically wherein the _EC50 is less than 50 μg/mL and more specifically less than 40 μg/mL, or wherein the K as measured by surface plasmon resonance (SPR) (usually at 25°C) or Luminex analysis (usually as described in the Examples) or Octet® ( _Abdiche et al. 2008) is 10 nM or less, and/or it has an affinity for binding BTNL8 relative to nonspecific binding of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratios; and/ or ii. it binds to the human BTNL8/BTNL3 dimer as expressed in a cell line, such as preferably in HEK293T cells stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. it does not bind to express BTNL3 A cell line other than BTNL8, such as the HEK-293T cell line expressing BTNL3, for example as described in the Examples; iv. It modulates the activation of T cells carrying the Vγ4Vδ1 TCR, typically as determined by the Vγ4Vδ1 TCR reporter cell assay ( For example as illustrated in the Examples), typically: • It inhibits activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 below 1 nM (usually between 0.1 and 1 nM), or below 0.2 µg/ml (usually in the between 0.01 and 0.2 µg/ml), as measured by the NF-AT-GFP gene reporter as a readout, it inhibits the activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 below 1.1 nM, typically at 0.3 between 1.1 nM or between 0.05 and 0.17 µg/ml, as measured by CD69 expression at the plasma membrane as a readout, and/or it inhibits TCR downregulation in T cells bearing Vγ4Vδ1 TCRs with an EC ₅₀ Below 1.4, typically between 0.5 and 1.4 nM or between 0.05 and 0.21 µg/ml, as measured by TCRVδ1 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits activated The lytic function of Vδ1 T cells, which generally inhibit the degranulation of Vδ1 T cells against HL-60 cells, as determined in an in vitro assay of degranulated cells (eg, as described in the Examples); vi. its inhibition from activated Vδ1 Interferon (eg: TNFα) production by T cells, generally as described in the examples and/or vii. it cross-reacts with cynomolgus monkey BTNL8, viii. it binds to human BTNL8, in particular to as expressed in a cell line, e.g. stably transduced with a lentiviral vector encoding human BTNL8 Human _BTNL8 in HEK293T cells (as described in the Examples), more specifically with a KD below 10 nM, in particular below 1 nM, as measured by BLI (generally as described in the Examples).

In various embodiments, the anti-BTNL8 antibodies of the invention can exhibit one, two, three, four, five, six, seven, or all of the desired functional properties discussed above. The anti-BTNL8 antibody can be, for example, a human antibody, a humanized antibody, or a chimeric antibody. Preferably, the antibody is a humanized or human antibody, more preferably a humanized silent antibody.

As used herein, the term "silencing" antibody refers to an antibody that exhibits no or low ADCC activity, as measured in an in vitro ADCC activity assay that measures cytolysis of target cells.

In one embodiment, the term "no ADCC activity or low ADCC activity" means that a silent antibody exhibits lower ADCC than is observed in the case of a corresponding wild-type (non-silent) antibody, eg, a wild-type human IgG1 antibody ADCC activity of 50% of the activity (eg, less than 10%). Preferably, no detectable ADCC activity is observed in the in vitro ADCC activity assay using the silent antibody compared to the control Fab antibody.

Silencing effector functions can be obtained by mutation in the Fc constant portion of the antibody and have been described in the art: Strohl 2009 (LALA &N297A); Baudino 2008, D265A (Baudino et al, J. Immunol. 181 (2008) ): 6664-69, Strohl, CO Biotechnology 20 (2009): 685-91). Examples of silencing IgGl antibodies include mutations at positions 234, 235 and/or 331 in the IgGl Fc amino acid sequence (EU numbering) that reduce ADCC. Another silent IgG1 antibody contained the N297A mutation, which resulted in deglycosylated or aglycosylated antibodies.

In some embodiments, the anti-BTNL8 antibodies of the invention are selected from the group consisting of Fab, F(ab')2, Fab' and scFv. As used herein, the term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, wherein in the fragment obtained by treating IgG with the protease papain, about half of the N-terminal side of the H chain and The entire L chain is held together via disulfide bonds. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity that is slightly larger than the disulfide via the hinge region in the fragment obtained by treating IgG with the protease pepsin Bonded Fab. The term "Fab'" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleavage of disulfide bonds in the hinge region of F(ab')2. Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers typically expressed by gene fusions comprising VH and VL encoding genes linked by peptide-encoding linkers. Human scFv fragments of the present invention include CDRs maintained in an appropriate configuration, preferably by using genetic recombination techniques.

Reference Antibodies mAbs X1 to X5 Antibodies of the invention include reference murine monoclonal anti-BTNL8 antibodies isolated and structurally characterized by their variable heavy and light chain amino acid sequences as described in Table 1 below.

In a preferred embodiment, the reference murine monoclonal antibody is mAb X1, mAb X2, mAb X4, mAb X5 rather than mAb X3. Table 1 : Variable heavy and light chain amino acid sequences of murine reference antibodies of the invention antibody VH amino acid sequence VL amino acid sequence mAb X1 SEQ ID NO: 1 SEQ ID NO: 2 mAb X2 SEQ ID NO: 3 SEQ ID NO: 4 mAb X3 SEQ ID NO: 5 SEQ ID NO: 6 mAb X4 SEQ ID NO: 7 SEQ ID NO: 8 mAb X5 SEQ ID NO: 9 SEQ ID NO: 10

VH CDR1 (also known as HCDR1), VH CDR2 (also known as HCDR2), VH CDR3 (also known as HCDR3), VL CDR1 (also known as LCDR1), VL CDR2 (also known as LCDR2) of some antibodies according to the invention ), an example of the amino acid sequence of VL CDR3 (also known as LCDR3) is shown in Table 2.

In Table 2, the CDR regions of some of the antibodies of the invention were delineated using the Kabat system.

For ease of reading, the CDR regions are hereinafter referred to as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively. Table 2 : CDR regions of reference murine antibodies as defined by Kabat original antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 mAb X1 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 mAb X2 SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 mAb X3 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 mAb X4 SEQ ID NO: 29 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 mAb X5 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40

In particular embodiments, isolated anti-BTNL8 antibodies according to the present invention comprise: (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, HCDR3 of SEQ ID NO:13, and LCDR1 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 16; (b) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:17, HCDR2 of SEQ ID NO:18, HCDR3 of SEQ ID NO:19, and LCDR1 of SEQ ID NO:20, HCDR1 of SEQ ID NO:21 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 22; (c) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:23, HCDR2 of SEQ ID NO:24, HCDR3 of SEQ ID NO:25, and LCDR1 of SEQ ID NO:26, HCDR1 of SEQ ID NO:27 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 28; (d) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:29, HCDR2 of SEQ ID NO:30, HCDR3 of SEQ ID NO:31, and LCDR1 of SEQ ID NO:32, HCDR3 of SEQ ID NO:33 LCDR2 and a variable light chain polypeptide of LCDR3 of SEQ ID NO: 34; or (e) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:35, HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, and LCDR1 of SEQ ID NO:38, HCDR1 of SEQ ID NO:39 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 40; Wherein the anti-BTNL8 antibody is specific for BTNL8.

In a preferred embodiment, the isolated anti-BTNL8 antibody according to the present invention is specific for BTNL8 and comprises: (a) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:11, HCDR2 of SEQ ID NO:12, HCDR3 of SEQ ID NO:13, and LCDR1 of SEQ ID NO:14, HCDR3 of SEQ ID NO:15 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 16; (b) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:17, HCDR2 of SEQ ID NO:18, HCDR3 of SEQ ID NO:19, and LCDR1 of SEQ ID NO:20, HCDR1 of SEQ ID NO:21 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 22; (c) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:29, HCDR2 of SEQ ID NO:30, HCDR3 of SEQ ID NO:31, and LCDR1 comprising SEQ ID NO:32, HCDR1 of SEQ ID NO:33 LCDR2 and a variable light chain polypeptide of LCDR3 of SEQ ID NO: 34; or (d) a variable heavy chain polypeptide comprising HCDR1 of SEQ ID NO:35, HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, and LCDR1 of SEQ ID NO:38, HCDR1 of SEQ ID NO:39 Variable light chain polypeptides of LCDR2 and LCDR3 of SEQ ID NO: 40;

In other specific embodiments, isolated anti-BTNL8 antibodies according to the present invention comprise: (a) a variable heavy chain polypeptide comprising the VH of SEQ ID NO: 1 and a variable light chain polypeptide comprising the VL of SEQ ID NO: 2; (b) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:3 and a variable light chain polypeptide comprising the VL of SEQ ID NO:4; (c) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:5 and a variable light chain polypeptide comprising the VL of SEQ ID NO:6; (d) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:7 and a variable light chain polypeptide comprising the VL of SEQ ID NO:8; or (e) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:9 and a variable light chain polypeptide comprising the VL of SEQ ID NO:10; Wherein the anti-BTNL8 antibody is specific for BTNL8.

In another preferred embodiment, the isolated anti-BTNL8 antibody according to the present invention is specific for BTNL8 and comprises: (a) a variable heavy chain polypeptide comprising the VH of SEQ ID NO: 1 and a variable light chain polypeptide comprising the VL of SEQ ID NO: 2; (b) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:3 and a variable light chain polypeptide comprising the VL of SEQ ID NO:4; (c) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:7 and a variable light chain polypeptide comprising the VL of SEQ ID NO:8; or (d) a variable heavy chain polypeptide comprising the VH of SEQ ID NO:9 and a variable light chain polypeptide comprising the VL of SEQ ID NO:10; Wherein the anti-BTNL8 antibody is specific for BTNL8.

Functionally Variant Antibodies In yet another embodiment, functionally variant antibodies of the invention have full-length heavy and light chain amino acid sequences; or variable region heavy and light chain amino acid sequences, or the same In Tables 1 and 2) the corresponding amino acid sequences of the reference antibody mAbs X1, X2, X3, X4 or X5 are homologous or more specifically identical to all 6 CDR region amino acid sequences, and wherein this The functionally variant-like antibodies exhibit the desired functional properties of any of the reference antibodies mAb X1 to mAb X5.

Functional variants of the reference mAbs X1 to X5 antibodies (especially functional variants of the VL, VH or CDRs used in the context of the monoclonal antibodies of the invention) still allow the antibody to retain affinity (usually as measured by Luminex assays) at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%), and/or parental antibodies (e.g., mAb _X1 to mAb X5 antibodies) the specificity/selectivity of any one), and in some cases, this monoclonal antibody of the invention may be more specific than the parental Ab (eg: any of mAb X1 to mAb X5 antibodies) Affinity, selectivity and/or specificity are related.

Desired functional properties of reference mAbs X1 to mAb X5 may be selected from the group consisting of: i. They are specific for BTNL8, in particular binding to cell lines as expressed, eg, stably transduced with a lentiviral vector encoding human BTNL8 Human BTNL8 in HEK293T cells (as described in the Examples), more specifically wherein the _EC50 is less than 50 μg/mL and more specifically less than 40 μg/mL, or wherein such as by surface plasmon resonance (SPR) (usually at 25°C) or Luminex assays (usually as described in the Examples) or Octet® (Abdiche et al. 2008) with a _K of 10 nM or less, and/or its ability to bind BTNL8 Has an affinity of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater relative to nonspecific binding; and/or ii. it binds to as expressed in cells strains, such as preferably human BTNL8/BTNL3 in HEK293T cells stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. It does not bind to cell strains expressing BTNL3 but not BTNL8, such as HEK- The 293T cell line, e.g., as described in the Examples; iv. It modulates activation of T cells carrying Vγ4Vδ1 TCR, typically as determined by a Vγ4Vδ1 TCR reporter cell assay (e.g., as described in the Examples), generally: It inhibits Vγ4Vδ1 carrying Activation of T cells by TCR with an _IC50 below 1 nM (usually between 0.1 and 1 nM), or below 0.2 µg/ml (usually between 0.01 and 0.2 µg/ml), as by NF- The AT-GFP gene reporter, measured as a readout, inhibits the activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 below 1.1 nM (usually between 0.3 and 1.1 nM), or below 0.17 µg/ml ( Typically between 0.05 and 0.17 µg/ml), as measured by CD69 expression at the plasma membrane as a readout, and/or it inhibits TCR downregulation in Vγ4Vδ1 TCR-bearing T cells with an _EC50 below 1.4 ( Typically between 0.5 and 1.4 nM), or below 0.21 µg/ml (typically between 0.05 and 0.21 µg/ml), as measured by TCRVδ1 expression at the plasma membrane as a readout, as illustrated in the Examples v. It inhibits the lytic function of activated Vδ1 T cells, typically it inhibits the degranulation of Vδ1 T cells against HL-60 cells, as determined in an in vitro assay of degranulated cells (eg, as described in the Examples); vi. . It is inhibited by activated Vδ1 T Cellular production of cytokines (eg: TNFα), generally assessed as described in the Examples, and/or vii. It cross-reacts with cynomolgus monkey BTNL8, viii. It binds to human BTNL8, specifically to as expressed Human BTNL8 in cell lines, such as HEK293T cells stably transduced with a lentiviral vector encoding human _BTNL8 (as described in the Examples), more specifically with a KD below 10 nM, specifically below 1 nM , as measured by BLI (generally as described in the Examples).

For example, the present invention relates to functionally variant antibodies of mAb X1 to mAb X5, preferably functionally variant antibodies of mAbs X1, X2, X4 and X5, comprising a variable heavy chain ( _VH ) and a variable light chain ( _VL ) sequence, wherein the CDR sequences (i.e., the 6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) and the corresponding CDR sequences of at least one antibody of mAb X1 to mAb X5 (as shown in Table 2 ) share at least 60, 70, 90, 95 or 100% sequence identity, wherein the functional variant antibody specifically binds to BTNL8, and the antibody exhibits at least one of the following functional properties: i. It is specific for BTNL8, specific In other words binding to human BTNL8 (as described in the Examples) as expressed in a cell line, such as HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, more specifically wherein the _EC50 is below 50 μg/mL and more specifically below 40 μg/mL, or wherein as analyzed by surface plasmon resonance (SPR) (usually at 25°C) or Luminex analysis (usually as described in the Examples) or Octet® (Abdiche et al. 2008) has a measured _K of 10 nM or less, and/or has about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity; ii. it binds to the human BTNL8/BTNL3 dimer as expressed in cell lines, such as preferably HEK293T cells stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3 , iii. it does not bind to cell lines expressing BTNL3 but not BTNL8, such as the HEK-293T cell line expressing BTNL3, for example as described in the Examples; iv. it modulates the activation of T cells bearing the Vγ4Vδ1 TCR, typically as by As determined by the Vγ4Vδ1 TCR reporter cell assay (eg, as described in the Examples), generally: It inhibits the activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 of less than 1 nM (usually between 0.1 and 1 nM), or Below 0.2 µg/ml (usually between 0.01 and 0.2 µg/ml), as measured by the NF-AT-GFP gene reporter as readout, it inhibits the activation of T cells carrying the Vγ4Vδ1 TCR, where IC ₅₀ Below 1.1 nM (usually between 0.3 and 1.1 nM), or below 0.17 µg/ml (usually between 0.05 and 0.17 µg/ml) as measured by CD69 expression at the plasma membrane as a readout , and/or ● which inhibits Vγ4Vδ1 T TCR downregulation of T cells in CR with _EC50 below 1.4 (usually between 0.5 and 1.4 nM), or below 0.21 µg/ml (usually between 0.05 and 0.21 µg/ml), e.g. by plasma membrane TCRVδ1 expression was measured as a readout, as described in the Examples; v. it inhibits the lytic function of activated Vδ1 T cells, typically it inhibits the degranulation of Vδ1 T cells against HL-60 cells, such as in vitro degranulation As determined in a cellular assay (e.g. as described in the Examples); vi. its inhibition of interferon (e.g.: TNFα) production from activated Vδ1 T cells, generally assessed as described in the Examples, vii. Cynomolgus BTNL8 cross-reacts, and/or viii. it binds to human BTNL8, in particular to human BTNL8 as expressed in cell lines, such as HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8 (as in the examples described), more specifically wherein the KD is below 10 nM, in particular below 1 nM, as measured by BLI (generally as described in the Examples).

The present invention further relates to functionally variant antibodies of mAb X1 to mAb X5, preferably functionally variant antibodies of mAbs X1, X2, X4 and X5, comprising a heavy chain corresponding to any of mAb X1 to mAb X5 antibodies and Light chain variable regions (as shown inter alia in Table 1) heavy chain variable regions and light chain variable regions at least 80%, 90%, or at least 95% or 100% identical; functional variant antibodies specifically bind to BTNL8, and exhibits at least one of the following functional properties: i. It is specific for BTNL8, in particular binding to HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8 as expressed in a cell line such as human BTNL8 as described in the Examples), more specifically wherein the _EC50 is less than 50 μg/mL and more specifically less than 40 μg/mL, or wherein such as by surface plasmon resonance (SPR) (typically at 25°C) or a Luminex assay (generally as described in the Examples) or Octet® (Abdiche et al. 2008) with a _K of 10 nM or less, and/or its binding to BTNL8 relative to nonspecific Binds with an affinity of about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratios; ii. it binds to a cell line as expressed, eg, preferably encoded human Human BTNL8/BTNL3 in HEK293T cells stably transduced with lentiviral vectors of BTNL8 and human BTNL3, iii. It does not bind to cell lines expressing BTNL3 but not BTNL8, such as the HEK-293T cell line expressing BTNL3, such as in the Examples described; iv. it modulates the activation of T cells bearing the Vγ4Vδ1 TCR, typically as determined by a Vγ4Vδ1 TCR reporter cell assay (eg, as described in the Examples), generally: It inhibits the activation of T cells bearing the Vγ4Vδ1 TCR, where the _IC50 is less than 1 nM (usually between 0.1 and 1 nM), or less than 0.2 µg/ml (usually between 0.01 and 0.2 µg/ml), as indicated by the NF-AT-GFP gene reporter as As measured by readout, it inhibits activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 below 1.1 nM (usually between 0.3 and 1.1 nM), or below 0.17 µg/ml (usually between 0.05 and 0.17 µg/ml) ml), as measured by CD69 expression at the plasma membrane as a readout, and/or it inhibits TCR downregulation in T cells bearing the Vγ4Vδ1 TCR with an _EC50 below 1.4 (usually between 0.5 and 1.4 nM between 0.21 µg/ml (usually between 0.05 and 0.21 µg /ml), as measured by TCRVδ1 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the lytic function of activated Vδ1 T cells, generally it inhibits Vδ1 T cells against HL- 60 Degranulation of cells, as determined in an in vitro degranulation cell assay (eg, as described in the Examples); vi. It inhibits the production of interferons (eg: TNFα) from activated Vδ1 T cells, generally as described in the Examples Evaluated as described, vii. it cross-reacts with cynomolgus monkey BTNL8, and/or viii. it binds to human BTNL8, in particular to as expressed in a cell line, eg, stably transduced with a lentiviral vector encoding human BTNL8 Human BTNL8 in HEK293T cells (as described in the Examples), more specifically with a K _D below 10 nM, in particular below 1 nM, as measured by BLI (generally as described in the Examples) .

The sequences of the CDR variants may differ from the sequences of the CDRs of the parent/reference antibody sequence (as eg shown in Table 2) primarily by conservative substitutions; eg at least 10 of the variants (such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1) substitutions are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions can be defined by substitutions within the amino acid class as reflected below: Aliphatic residues I, L, V and M Cycloalkenyl related residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Minor residues A, C, D, G, N, P, S, T and V Minimal residues A, G and S A, C, D, E, G, H, K, N, Q, R, S, P and residues forming T are involved in this order Flexible residues Q, T, K, S, G, P, D, E and R

More conservative substitution groups include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and aspartic-glutamic acid . Conservation with respect to hydrophilic/hydrophilic character and residue weight/size is also substantially preserved in the variant CDRs compared to the CDRs of any of mAbs X1 to X5.

The importance of the hydrophilic amino acid index in conferring interactive biological function on proteins is generally understood in the art. It is recognized that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like) . Each amino acid has been assigned a hydrophilicity index based on its hydrophobicity and charge characteristics, these amino acids are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); amphetamine acid (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7) ; Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Aspartic acid (-3.5); aspartic acid (-3.5); aspartic acid (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues can also or alternatively be measured by similarity scores, such as by using a BLAST program (e.g. BLAST 2.2.8 available via NCBI, using standard settings BLOSUM62, Open Gap=11 and Extended Gap =1) determined.

Suitable variants typically exhibit at least about 90% (eg, 95%) identity to the parent polypeptide VH and VL sequences. According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence and the second amino acid sequence have 90%; 91%; 92%; 93%; 94 %; 95%; 96%; 97%; 98%; 99%; or 100% agreement. According to the invention, a first amino acid sequence having at least 50% identity with a second amino acid sequence means that the first sequence and the second amino acid sequence have 50%; 51%; 52%; 53%; 54 %; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%; 69%; 70%; 71%; 72%; 73%; 74%; 75%; 76%; 77%; 78%; 79%; 80%; 81%; 82%; 83%; 84%; 85%; 86%; 87% 88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; or 100% agreement.

In some embodiments, the functional variant is a chimeric antibody, typically a chimeric mouse/human antibody. The term "chimeric antibody" refers to a monoclonal antibody comprising the VH and VL domains of an antibody derived from a non-human animal, the CH and CL domains of a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. In particular, the mouse/human chimeric antibody can comprise the VH and VL domains of any of the mAb X1 to mAb X5 reference antibodies.

In some embodiments, the functional variant is a humanized antibody. In particular embodiments, the antibodies of the invention are humanized antibodies comprising the 6 CDRs of any of the mAb X1 to mAb X5 reference antibodies, eg, as shown in Table 2. As used herein, the term "humanized antibody" refers to an antibody in which the framework regions (FRs) have been modified to avoid potentially immunological residues in humans. For example, a humanized antibody system comprises the FRs of a donor immunoglobulin from a human species compared to the FRs of a parental immunoglobulin (eg, murine CDRs).

Functionally variant antibodies with mutated amino acid sequences can be obtained by mutagenesis (eg, site-directed mutagenesis or PCR-mediated mutagenesis) of the encoding nucleic acid molecule, and then tested for the encoded nucleic acid molecules using functional assays described herein. The altered antibody retains the function (ie, the function described above).

Antibodies that cross-compete at least one of mAb X1 to mAb X5 and / or bind to the same epitope as one of mAb X1 to mAb X5 in a standard BTNL8 binding assay, as disclosed herein with reference antibodies mAb X1 to Additional antibodies of similar favorable properties of at least one of mAb X5 can cross-compete (eg, competitively inhibit binding thereof) in a statistically significant manner with any of the reference antibodies mAb X1 to mAb X5 as described above. ability to identify.

Test antibodies can be first screened for their binding affinity to BTNL8, eg, from human recombinant antibody libraries using, eg, phage display technology, or from transgenic mice expressing human variable region antibodies immunized with the BTNL8 antigen.

The ability of the test antibody to cross-compete with the antibody of the invention or inhibit the binding of the antibody of the invention to human BTNL8 confirms that the test antibody can compete with the antibody for binding to human BTNL8; according to a non-limiting theory, the antibody can bind to the antibody it competes with to the same or related (eg, structurally similar or spatially adjacent) epitope to human BTNL8.

To screen an anti-BTNL8 antibody for its ability to bind to the same epitope as one of the mAb X1 to mAb X5 reference antibodies, for example, HEK293 cells transfected with human BTNL8 and human BTNL3 (as described in the Examples) were Staining with one of the reference antibodies mAb X1 to mAb X5 at a saturating concentration (10 μg/mL) during 30 minutes at 4°C. After 2 washes, different doses of the test anti-BTNL8 mAbs were tested for their potential to compete with any of the mAb X1 to mAb X5 reference antibodies (30 min at 4°C). Indeed a mAb that competes with the reference antibody for the same binding site will not be able to recognize BTNL8 in the presence of such a reference antibody. Data can be expressed as mean fluorescence intensity.

Selected antibodies can be further tested for favorable properties of mAb X1 to mAb X5, in particular with respect to inhibitory properties against activated Vγ4Vδ1 T cells.

Accordingly, in one embodiment, the present invention provides an isolated antibody that competes for binding to at least one reference antibody of mAb X1 to mAb X5 from binding to BTNL8, wherein the antibody exhibits at least one of the following properties: i . It is specific for BTNL8, specifically binding to human BTNL8 as expressed in cell lines, such as HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8 (as described in the Examples), more specifically wherein the _EC50 is below 50 μg/mL and more specifically below 40 μg/mL, or wherein as by surface plasmon resonance (SPR) (usually at 25°C) or Luminex analysis (usually as in the Examples) Description) or Octet® ( _Abdiche et al. 2008) with a KD of 10 nM or less, and/or it has about 10:1, about 20:1, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratios of affinity; and/or ii. its binding to cell lines as expressed, for example, preferably stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3 Human BTNL8/BTNL3 dimer in HEK293T cells, iii. it does not bind to cell lines expressing BTNL3 but not BTNL8, such as HEK-293T cell lines expressing BTNL3, e.g. as described in the Examples; iv. Activation of T cells with the Vγ4Vδ1 TCR, typically as determined by a Vγ4Vδ1 TCR reporter cell assay (eg, as described in the Examples), typically: It inhibits the activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 of less than 1 nM (usually between 0.1 and 1 nM), or below 0.2 µg/ml (usually between 0.01 and 0.2 µg/ml), as measured by the NF-AT-GFP gene reporter as a readout, which Inhibits activation of Vγ4Vδ1 TCR-bearing T cells with an _IC50 below 1.1 nM (usually between 0.3 and 1.1 nM), or below 0.17 µg/ml (usually between 0.05 and 0.17 µg/ml), as indicated by As measured by CD69 expression at the plasma membrane as a readout, and/or it inhibits TCR downregulation in T cells bearing the Vγ4Vδ1 TCR with an _EC50 below 1.4 (usually between 0.5 and 1.4 nM), or below 0.21 µg/ml (usually between 0.05 and 0.21 µg/ml), as measured by TCRVδ1 expression at the plasma membrane as a readout, as illustrated in the Examples; v. It inhibits the lytic function of activated Vδ1 T cells , which normally inhibits Vδ1 T cells against HL-60 cells Degranulation, as determined in an in vitro assay of degranulated cells (eg, as described in the Examples); vi. It inhibits interferon (eg: TNFα) production from activated Vδ1 T cells, typically as described in the Examples Assessing, vii. its binding to human BTNL8, in particular to human BTNL8 as expressed in a cell line, such as HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8 (as described in the Examples), more specifically wherein the KD is below 10 nM, in particular below 1 nM, as measured by BLI (usually as described in the Examples), and/or viii. It cross-reacts with cynomolgus BTNL8.

Typically, the functional properties according to points (iv) to (viii) above of an antibody that competes with at least one of the reference mAbs X1 to X5 for binding to BTNL8 are substantially equal to or better than the corresponding reference antibody mAb X1 as described above to the corresponding functional characteristics of X5. Substantially equivalent is intended herein that functional variants retain at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of the corresponding functional properties of the reference mAbs X1 to X5.

Typically, an antibody that competes with any of the reference mAbs X1 to X5 according to the invention for binding to BTNL8 still has at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%) of the affinity of the reference antibody , 95%, or 100%), and in some cases may be associated with greater affinity, selectivity, and/or specificity than the reference antibody.

In another embodiment, the present invention provides antibodies that bind to the same epitope as at least one of the anti-BTNL8 antibodies mAb X1 to mAb X5 as described herein.

In a specific embodiment, the present invention provides antibodies, preferably chimeric, humanized or human recombinant antibodies, that bind or cross-compete to the same epitope as anti-BTNL8 antibody mAb X1 and do not bind to anti-BTNL8 antibody mAb X2 , X4 and X5 bind or cross-compete to the same epitope.

In a specific embodiment, the present invention provides antibodies, preferably chimeric, humanized or human recombinant antibodies, that bind or cross-compete to the same epitope as the anti-BTNL8 antibody mAbs X2, X4 and X5, and that do not bind to the anti-BTNL8 antibody mAbs X2, X4 and X5. BTNL8 antibody mAb X1 binds or cross-competes to the same epitope.

In a certain embodiment, the one or more cross-competing antibodies that bind to the same epitope on human BTNL8 as any of mAb X1 to mAb X5 are chimeric, humanized, or human recombinant antibodies.

Generation of Monoclonal Antibody-Producing Metastases The antibody systems of the invention are produced by any technique known in the art such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination. Generally, the amino acid sequence of the desired sequence is known, and those skilled in the art can readily generate such antibodies by standard techniques for the production of polypeptides. For example, the antibodies can be synthesized using well known solid phase methods, preferably using commercially available peptide synthesis equipment such as those of Applied Biosystems, Foster City, California, and following the manufacturer's instructions.

Alternatively, the antibodies of the invention can be synthesized by recombinant DNA techniques well known in the art. For example, antibodies can be obtained as DNA expression products following incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts, which hosts will then express themselves from The desired antibody for its isolation.

Therefore, another object of the present invention relates to a nucleic acid molecule encoding an antibody according to the invention, typically the reference antibody mAbs X1 to X5 or functional variants thereof. More specifically, the nucleic acid molecule encodes the heavy or light chain of an antibody of the invention. More specifically, the nucleic acid molecule comprises at least 70%, 80% of the corresponding nucleic acid encoding the heavy chain variable region (VH region) or light chain variable region (VL) of any of the reference antibodies mAb X1 to mAb X5. %, 90%, 95% or 100% identical VH or VL coding regions, for example as SEQ ID NO 47-48 (mAb X1), SEQ ID NO: 55-56 (mAb X2), SEQ ID NO: 63- 64 (mAb X3), SEQ ID NOs: 71-72 (mAb X4), SEQ ID NOs: 79-80 (mAb X5).

Typically, the nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plastid, cosmid, episomal, artificial chromosome, bacteriophage or viral vector. As used herein, the terms "vector", "colonization vector" and "expression vector" mean that DNA or RNA sequences (eg, foreign genes) can be introduced into a host cell in order to transform the host and facilitate the incorporation of the introduced sequences. Mediators of expression (eg, transcription and translation). Therefore, another object of the present invention relates to a vector comprising the nucleic acid of the present invention for producing antibodies. Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, and the like, to facilitate or direct the expression of the antibody after administration to an individual. Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40, the LTR promoter and enhancer of Moloney mouse leukemia virus, the promoter and enhancer of immunoglobulin H chain. Promoters and enhancers and the like. Any expression vector for animal cells can be used as long as the gene encoding the C region of a human antibody can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, and the like. Other examples of plastids include replicating or integrating plastids comprising an origin of replication, such as, for example, pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfection of encapsulated cells or by transient transfection with helper plastids or viruses. Typical examples of virus-encapsulating cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and the like. Detailed protocols for the production of such replication deficient recombinant viruses can be found, for example, in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.

Another object of the present invention relates to a host cell transfected, infected or transformed by the nucleic acid and/or vector as described above. As used herein, the term "transformation" means introducing a "foreign" (ie, foreign or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce the desired substance , usually the antibody encoded by the introduced gene or sequence. A host cell that accepts and expresses the introduced DNA or RNA has been "transformed".

Nucleic acids of the present invention can be used to generate antibodies of the present invention in suitable expression systems. The term "expression system" means a host cell and a compatible vector under suitable conditions, eg, for the expression of proteins encoded by exogenous DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plastid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyce or Saccharomyces yeast, mammalian cell lines (eg, Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established of mammalian cell cultures (eg, produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene (hereinafter "DHFR gene") is deficient (Urlaub G et al.; 1980), rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter "YB2/0 cells") and the like.

The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, the method comprising the steps of: (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo , (ii) recombinant host cells obtained by in vitro or ex vivo culture and (iii) optionally selected cells expressing and/or secreting the antibody. Such recombinant host cells can be used to produce the antibodies of the present invention. The recombinant nucleic acid can be stably integrated into the genome of the host cell.

Antibodies of the invention are suitably isolated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

In some embodiments, human chimeric antibodies of the present invention can be produced by obtaining nucleic acid sequences encoding the VL and VH domains as previously described, by inserting them into a human antibody encoding human antibody CH and human antibody A human chimeric antibody expression vector was constructed by using the expression vector of the animal cell of the gene of CL, and the coding sequence was expressed by introducing the expression vector into the animal cell. As the CH domain of a human chimeric antibody, it can be any region belonging to human immunoglobulins, but those of the IgG class are suitable, and children belonging to the IgG class such as IgG1, IgG2, IgG3 and IgG4 can also be used any of the classes. In addition, as the CL of the human chimeric antibody, it can be any region belonging to the Ig, and those regions of the kappa class or the lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques well known in the art (see Morrison SL. et al. (1984) and patent documents US 5,202,238; and US 5,204,244).

Humanized antibodies of the invention can be produced by obtaining nucleic acid sequences encoding CDR domains as previously described, and constructing humanized antibody expression vectors by inserting them into expression vectors having genes encoding: (i ) heavy chain constant regions and heavy chain variable framework regions identical to the heavy chain constant regions and heavy chain variable framework regions of human antibodies and (ii) identical light chain constant regions and light chain variable framework regions of human antibodies light chain constant region and light chain variable framework region, and the gene is expressed by introducing the expression vector into a suitable cell line. Humanized antibody expression vectors can be of the type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or the type in which both genes are present on the same vector (tandem type).

Methods for humanizing antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, eg, Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91/09967; US Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), Veneer or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al (1994); Roguska MA et al (1994)) and chain shuffling (US Pat. No. 5,565,332). General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

The Fab of the present invention can be obtained by treating an antibody specifically reactive with AMH with the protease papain. Alternatively, Fabs can be produced by inserting DNA encoding the Fab of the antibody into a vector of a prokaryotic or eukaryotic expression system, and introducing the vector into a prokaryotic or eukaryotic organism, as appropriate, to express the Fab.

The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with AMH with the protease pepsin. Additionally, F(ab')2 can be generated by binding the Fab' described below via a thioether bond or a disulfide bond.

The Fab' of the present invention can be obtained by treating F(ab')2, which specifically reacts with AMH, with the reducing agent dithiothreitol. Alternatively, expression thereof can be performed by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism, as appropriate Fab' is produced.

The DNA encoding the scFv can be constructed by obtaining cDNA encoding the VH and VL domains as previously described, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into the prokaryotic or eukaryotic expression vector. The scFv of the present invention is produced by expressing the scFv in nuclear organisms (where appropriate).

To generate humanized scFv fragments, a well-known technique called CDR grafting can be used, which involves selecting complementarity determining regions (CDRs) from a donor scFv fragment and grafting them onto a human scFv fragment framework of known three-dimensional structure (see For example WO 98/45322; WO 87/02671; US 5,859,205; US 5,585,089; US 4,816,567; EP0173494).

Engineered antibodies of the invention further include those in which framework residues within VH and/or VL have been modified (eg, to improve the properties of the antibody). Such framework modifications are typically made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing antibody framework sequences to germline sequences from which the antibody is derived. To restore the framework region sequence to its germline configuration, somatic mutation can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention. Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in further detail in US Patent Publication No. 20030153043 to Carr et al.

Isotypes and Fc Engineering The constant regions of the antibodies of the invention may belong to any isotype.

As used herein, the terms "constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. A human IgG heavy chain Fc region is generally defined as comprising amino acid residues from position C226 or P230 of an IgG antibody to the carboxy terminus, wherein numbering is according to the EU numbering system. The C-terminal lysine (residue K447) of the Fc region can be removed, eg, during production or purification of the antibody or its corresponding codon deleted in the recombinant construct. Thus, the composition of the antibodies of the present invention may include antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations with mixtures of antibodies with or without K447 residues.

Isotype selection will typically be guided by the desired effector function, such as ADCC silencing. Exemplary isotypes are IgGl, IgG2, IgG3, and IgG4. Either human light chain constant region kappa or lambda can be used. If necessary, the class of the antibody of the present invention can be converted by known methods. Typical class switching techniques can be used to convert one IgG subclass to another, eg, from IgG1 to IgG2. Thus, the effector function of the antibodies of the invention can be altered by isotype switching to, for example, IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic uses. In some embodiments, the antibodies of the invention are full-length antibodies. In some embodiments, the full-length antibody is an IgGl antibody.

Antibodies of the invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues such that the antibody has an altered affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The affinity-altering effector ligand can be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in US Patent Nos. 5,624,821 and 5,648,260 (both by Winter et al.).

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or eliminated complement dependence Cytotoxicity (CDC). This method is described in further detail in US Patent No. 6,194,551 to Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This method is further described in PCT Publication WO 94/29351 to Bodmer et al.

In other embodiments, the Fc region is modified by modifying one or more amino acids to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to reduce the affinity of the antibody for Fcγ receptors. Such antibodies with reduced effector function, and in particular reduced ADCC, include silent antibodies.

In certain embodiments, an Fc domain of the IgGl isotype is used. In some specific embodiments, mutant variants of IgG1 Fc fragments are used, eg, silent IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or bind to Fcγ receptors.

In certain embodiments, an Fc domain of the IgG4 isotype is used. In some specific embodiments, mutant variants of IgG4 Fc fragments are used, eg, silent IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or bind to Fcγ receptors.

Silenced effector functions can be obtained by mutation in the Fc constant portion of the antibody and have been described in the art (Baudino et al., 2008; Strohl, 2009). An example of a silent IgG1 antibody includes the triple mutant variant IgG1 L247F L248E P350S. An example of a silent IgG4 antibody includes the double mutant variant IgG4 S241P L248E.

In certain embodiments, the Fc domain is a silent Fc mutation that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine at position 314. An example of such amino acid substitution is the substitution of N314 by glycine or alanine.

In some embodiments, the full-length anti-BTNL8 antibody of the present invention is an IgG4 antibody. In some embodiments, the anti-BTNL8 antibody is a stable IgG4 antibody. An example of a suitable stable IgG4 antibody is an antibody in which arginine at position 409 in the heavy chain constant region of human IgG4 (as indicated in Kabat et al. (supra)) is converted to lysine, threonine , methionine or leucine, preferably lysine (described in WO2006033386) substituted, and/or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stable IgG4 antibodies are disclosed in WO2008145142.

In some embodiments, the antibodies of the invention do not comprise an Fc moiety that induces antibody-dependent cellular cytotoxicity (ADCC). The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, such as about amino acids (aa) 230 to about aa 450 of a human gamma heavy chain or its counterpart in other types of antibodies. Corresponding sequences in heavy chains (eg, alpha, delta, epsilon, and mu of human antibodies), or their naturally occurring allotypes. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this invention (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

In some embodiments, the antibodies of the invention do not comprise an Fc domain capable of substantially binding to a FcyRIIIA (CD16) polypeptide. In some embodiments, the antibodies of the invention lack an Fc domain (eg, lack a CH2 and/or CH3 domain), or comprise an Fc domain of the IgG2 or IgG4 isotype. In some embodiments, the antibodies of the invention consist of or comprise: Fab, Fab', Fab'-SH, F(ab')2, Fv, diabodies, single chain antibody fragments or comprise a plurality of different antibody fragments of multispecific antibodies.

In some embodiments, the antibodies of the invention are not linked to toxic moieties. In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered C2q binding and/or reduced or eliminated complement-dependent cells Toxicity (CDC). This method is described in further detail in US Patent No. 6,194,551.

In addition, the antibodies of the invention can be chemically modified (eg, one or more chemical moieties can be attached to the antibody), or modified to alter their glycosylation, which in turn alters one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

Modifications of the antibodies herein encompassed by the present invention are pegylation or polyhydroxyethyl starchylation or related techniques. Antibodies can be pegylated, eg, to increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically combined with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, before linking one or more PEG groups to the antibody or antibody fragment. reaction under conditions. Pegylation can be carried out by acylation or alkylation with reactive PEG molecules (or similarly reactive water-soluble polymers). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy-polyethylene glycol or aryloxy-polyethylene glycol Diol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is a deglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the invention. See, eg, EP 0154 316 to Nishimura et al. and EP 0 401 384 to Ishikawa et al.

Another modification to antibodies encompassed by the present invention is the binding or protein fusion of at least the antigen binding region of the antibodies of the present invention to a serum protein, such as human serum albumin or a fragment thereof, to increase the half-life of the resulting molecule.

In some embodiments, the invention also provides a multispecific antibody. Exemplary versions of multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one specific for BTNL8 and the other for a second antigen specificity; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, such as two scFvs linked in tandem by additional peptide linkers; (iv) dual Variable single domain antibodies (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem via short peptide bonds (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) ) Molecule, in: Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandab, two of which produce tetravalent bispecific antibodies Fusions of single chain diabodies, the tetravalent bispecific antibodies having two binding sites for each of the target antigens; (vii) flexibodies, which are the interaction between the scFv and the generation of multivalent molecules Combinations of diabodies; (viii) so-called "dock and lock" molecules, which are based on the "dimerization and docking domains" in protein kinase A, which, when applied to Fab, result in Trivalent bispecific binding proteins composed of two identical Fab fragments of different Fab fragments; (ix) so-called scorpions comprising, for example, two scFvs fused to the two ends of human Fab arms; and (x) double functional antibodies. Another exemplary format of bispecific antibodies is an IgG-like molecule with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known techniques such as those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain Body (SEED Body) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, bispecific antibodies are typically obtained or obtainable via controlled Fab arm exchange using DuoBody technology. In vitro methods for the production of bispecific antibodies by controlled Fab arm exchange have been described in WO2008119353 and WO 2011131746 (both by Genmab A/S). In an exemplary method described in WO 2008119353, when incubated under reducing conditions, bispecific antibodies are produced by a "Fab arm" or "Fab arm" between two monospecific antibodies (both comprising an IgG4-like CH3 region). Half-molecular "exchange" (exchange of heavy chain and attached light chain) is formed. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences. In another exemplary method described in WO 2011131746, the bispecific antibody system of the present invention is prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is an antibody of the present invention: a ) providing a first antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a second CH3 region; wherein the first The sequences of the first and second CH3 regions are different and such that the heterodimeric interaction between the first and second CH3 regions is stronger than each of the homodimeric interactions of the first and second CH3 regions; c ) incubating the primary antibody with the secondary antibody under reducing conditions; and d) obtaining the bispecific antibody, wherein the primary antibody is an antibody of the invention and the secondary antibody has a different binding specificity, or vice versa Of course. Reducing conditions can be provided, for example, by adding a reducing agent such as selected from 2-mercaptoethylamine, dithiothreitol, and gins(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to non-reducing or less reducing, eg by removing the reducing agent, eg by desalting. Preferably, the sequences of the first and second CH3 regions are different and contain only a few fairly conserved asymmetric mutations, so that the heterodimeric interaction between the first and second CH3 regions is stronger than that of the first and second CH3 regions Each of the homodimeric interactions of the CH3 region. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is incorporated herein by reference in its entirety. The following are illustrative examples of combinations of such asymmetric mutations, optionally where one or both Fc regions are of the IgGl isotype.

Pharmaceutical Compositions In another aspect, the present invention provides a composition, eg, a pharmaceutical composition, comprising at least one antibody as disclosed herein formulated together with a pharmaceutically acceptable carrier. Such compositions may include one antibody or a combination of (eg, two or more different) antibodies as described above. The pharmaceutical compositions disclosed herein may also be administered in combination therapy, ie, in combination with other agents.

For example, the antibodies of the invention can generally be combined with at least one antiviral, anti-inflammatory, or antimicrobial agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies of the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and its similar. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). In one embodiment, the carrier should be suitable for the subcutaneous route.

The formulations may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent loss of protein on the surface of the vial, and the like.

The pharmaceutical compositions of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for formulation capable of injection. Such vehicles can be, inter alia, isotonic sterile physiological saline solutions (mono- or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, In particular, freeze-dried compositions are permitted to constitute injectable solutions with the addition of sterile water or physiological saline as appropriate. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Thus, sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

The dosage for administration thereof can be adapted according to various parameters, and in particular according to the mode of administration used, the relevant pathology, or alternatively the desired duration of treatment.

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, and the like.

Suitable formulations of solutions for infusion or subcutaneous injection of antibodies have been described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4):519-530).

Methods of Using the Antibodies of the Present Invention The antibodies of the present invention have in vitro and in vivo diagnostic and therapeutic utility. For example, these molecules can be administered to cells in culture (eg, in vitro or in vivo) or in individuals (eg, in vivo) to treat, prevent, or diagnose a variety of disorders.

The anti-BTNL8 antibodies of the present invention are particularly useful in the treatment, prevention or diagnosis of BTNL8/BTNL3-related disorders or disorders associated with altered expression of BTNL8/BTNL3 or with Vδ1 T cells (specifically Vγ4Vδ1 T cells) in an individual in need thereof Use in a method for a disorder associated with an undesired activity, the method comprising administering to a subject a therapeutically effective amount of the anti-LTN8 antibody of the invention.

As used herein, a disorder associated with undesired activity of Vδ1 T cells (specifically Vγ4Vδ1 T cells) refers to the induction, enhancement or exacerbation in the presence of activated Vδ1 T cells (specifically Vγ4Vδ1 T cells) and /or any condition that can be treated by reducing the function of activated Vδ1 T cells. Such disorders include, inter alia, conditions associated with or characterized by abnormal expression of BTNL8 or BTNL8/BTNL3, and/or by modulating BTNL8 and/or BTNL8-induced signaling activity in human blood cells, such as by inhibiting A disease or condition that is treated by activated Vδ1 T cells for the production of cytokines and/or via the lytic function of activated Vδ1 T cells.

Therefore, the anti-BTNL8 antibodies of the present invention can be used in methods for inhibiting Vδ1 T cell activation, in particular in the presence of cells expressing BTNL8/BTNL3, in terms of interferon secretion or lysis function, such methods An inhibitory effective amount of the anti-BTNL8 antibody of the present invention is administered to an individual.

One object of the present invention therefore relates to a method of inhibiting an immune response in an individual, in particular inhibiting the lytic properties of Vγ4Vδ1 T cells in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-BTNL8 of the present invention antibody.

In particular embodiments, the anti-BTNL8 antibodies of the invention are particularly useful for treating, preventing or diagnosing inflammatory disorders, such as intestinal inflammation, in a subject.

The present invention also relates to a method of manufacturing a medicament for the treatment of an inflammatory condition, the medicament comprising an anti-BTNL8 antibody of the invention as described in the previous section.

Examples of treatable inflammatory diseases include, but are not limited to, inflammatory bowel disease, irritable bowel syndrome, diverticulitis, celiac disease, Crohn's disease, ulcerative colitis, thyroiditis, metabolic disorders, immune-related disorders, autoimmunity Disorders, transplant rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke, and infectious diseases.

In particular embodiments, the antibodies of the invention are used to treat disorders of the gastrointestinal system and/or associated with intestinal inflammation. Such disorders of the gastrointestinal system include, but are not limited to, inflammatory disorders of the gastrointestinal system and microbial infections of the tissues of the gastrointestinal system.

Antibodies of the invention can be administered as the sole active ingredient or in combination with other drugs (eg, immunosuppressive or immunomodulatory or other anti-inflammatory agents), such as as adjuvants to other drugs or in combination with other drugs, such as with For the treatment or prevention of the diseases mentioned above.

Typically, the anti-inflammatory agent may include, but is not limited to, an anti-inflammatory interleukin optionally selected from interleukin 10 (IL-10), interleukin 22 (IL-22). In other embodiments, the anti-inflammatory agent may include, but is not limited to, steroids, such as glucocorticoids, prednisone, hydrocortisone, or immunomodulators, including anti-TNFα antibodies or anti-IL -17 antibody.

As used herein, the term "treatment/treat" refers to both prophylactic or prophylactic treatment as well as curative or disease-modifying treatment, including treatment of individuals at risk of contracting a disease or suspected of having contracted a disease and Individuals who are sick or have been diagnosed with a disease or medical condition, and include suppression of clinical relapse. The treatment may be administered to an individual suffering from a medical condition or ultimately acquiring the condition in order to prevent, cure or relapse, delay the onset, reduce the severity or ameliorate one or more of its symptoms, or to improve the survival of the individual. prolongation of survival beyond what would be expected in the absence of this treatment.

As used herein, a "therapeutically effective amount" refers to an amount effective at a dose and for a period of time necessary to achieve the desired therapeutic result. A therapeutically effective amount of an antibody of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the antibody of the invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Effective doses and dosing regimens of the antibodies of the invention will depend on the disease or condition to be treated, and can be determined by those skilled in the art. A physician of ordinary skill in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, a physician may start a dose of an antibody of the invention employed in a pharmaceutical composition below the level necessary to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, a suitable dosage of the compositions of the present invention will be that amount of the compound that is the lowest dose effective to produce the therapeutic effect according to the particular dosing regimen. This effective dose will generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize disease progression. Typically, the ability of a compound to treat an inflammatory disorder is assessed, for example, in an animal model system that predicts efficacy in treating an inflammatory disorder. Alternatively, this property of a composition can be assessed by examining the ability of a compound to inhibit the induction of an immune response using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce an immune or inflammatory response or otherwise ameliorate symptoms in an individual. One of ordinary skill in the art will be able to determine such amounts based on factors such as the individual's size, the severity of the individual's symptoms, and the particular composition or route of administration chosen. An exemplary non-limiting range for a therapeutically effective amount of an antibody of the invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, eg, about 0.1-20 mg/kg, such as about 0.1-10 mg/kg , for example about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg, or about 8 mg/kg. An exemplary non-limiting range of a therapeutically effective amount of an antibody of the invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, such as about 0.5-2 mg/kg.

In accordance with the foregoing, the present invention provides in yet another aspect: A method as defined above comprising, for example, co-administering a therapeutically effective amount of an anti-BTNL8 antibody of the invention and at least one second drug substance, the second drug substance being an antiviral, anti-inflammatory or anti-viral agent, for example, simultaneously or sequentially Microbial agents, eg, as indicated above.

In one embodiment, the antibodies of the present invention can also be used to detect the levels of cells expressing BTNL8 or BTNL8/BTNL3. This can be done, for example, by incubating a sample (such as an ex vivo sample) and a control sample with an anti-BTNL8 antibody under conditions that allow the formation of a complex between the antibody and BTNL8 (such as expressed at the surface of a cell, such as in a blood sample) achieved. Any complexes formed between the antibody and BTNL8 are detected and compared in samples and controls. For example, standard detection methods well known in the art, such as ELISA and flow cytometry analysis, can be performed using the compositions of the present invention.

Accordingly, in one aspect, the present invention further provides methods for detecting the presence of BTNL8 or cells expressing BTNL8 (eg, human BTNL8 antigen) or measuring the amount of BTNL8 in a sample, comprising allowing the antibody to interact with The samples and control samples were incubated with an antibody of the invention that specifically binds to BTNL8 under conditions where complexes are formed between BTNL8. Complex formation is then detected, wherein differences in complex formation between the compared and control samples are indicative of the presence of BTNL8 in the samples.

In addition, kits and instructions for use consisting of the compositions disclosed herein (eg, the humanized antibodies of any of the reference antibodies mAbs X1 to X5) are within the scope of the invention. The kit may further contain at least one additional reagent, or one or more additional antibodies or proteins. The kit typically includes indicia indicating the intended use of the contents of the kit. The term indicia includes any written or recorded material on or supplied with the kit or otherwise accompanying the kit. The kit may further comprise means for diagnosing whether a patient belongs to a group that will respond to treatment with an anti-BTNL8 antibody as defined above.

The invention will be further illustrated by the following drawings and examples. However, these examples and drawings should not be construed in any way to limit the scope of the invention.

DESCRIPTION OF THE FIGURES Figure 1 : Identification of anti- BTNL8 mAbs . A. Screening cascade of anti-BTNL8 mAbs from mouse immunization to mAb sequencing. B. Bar graph showing the number of pure lines per affinity ( _KD ) range as measured on Luminex during primary hit selection. C. Stacked bar graph showing the number of anti-BTNL8 fusion tumor supernatant hits from primary screening that stained positive for the indicated cell line (MFI _fusion > MFI _HCM ) or negative (MFI _fusion < MFI _HCM ).

Figure 2. Anti- BTNL8 fusion tumor supernatant based screening of Vγ4Vδ1 TCR reporter cells. A. Sketch illustrating Vγ4Vδ1 TCR reporter assay in which JRT3-Vγ4Vδ1-202 reporter cells were combined with HEK-BTNL8/BTNL3 cells (ratio 1:1) with or without 50 µL of anti-BTNL8 or irrelevant fusion tumor supernatant (control). ) were co-cultured for 16 h, resulting in BTNL8/BTNL3-mediated activation, as depicted by up-regulation of CD69 and NF-AT-GFP reporters and down-regulation of TCRVδ1 at the plasma membrane of reporter cells. B. Dot plots (top) and histograms (middle and bottom) of flow cytometry modulating JRT3-Vγ4Vδ1-202 reporter cell activation in the presence of HEK-BTNL8/BTNL3 and anti-BTNL8 or control fusion tumor supernatants Examples of metrology patterns. C. Stacked bar graph showing changes in the expression of NF-AT-GFP reporter, CD69 and TCRVδ1 at the plasma membrane of JRT3-Vγ4Vδ1-202 reporter cells induced in the presence of 47 anti-BTNL8 fusion tumor supernatants , these anti-BTNL8 fusion tumor supernatants were found to modulate reporter activation during primary hit screening.

Figure 3. Anti- BTNL8 mAb enhances lysis of Vδ1 T cells . Vδ1 T cell lines were expanded from PBMCs of 3 healthy donors (see Materials and Methods) and grown in the presence of anti-CD107ab antibody and Golgistop with or without anti-BTNL8 or control fusion tumor supernatants Co-culture with HL-60 target cells using a 1:1 effector:target (E:T) ratio at 37°C. After 4 hours, cells were harvested, stained and analyzed on flow cytometry. A. BTNL8 expression in the HL-60 leukemia cell line as assessed on flow cytometry. B. Demonstrates degranulation of Vδ1 T cells in Vδ1 T cells cultured against HL60 myeloid leukemia cells alone or in the presence of a control isotype mAb (control) or anti-Vδ1 TCR mAb (positive control) or a representative reference anti-BTNL8 antibody ( % of CD107ab+ cells).

Figure 4. Anti- BTNL8 mAb inhibits activation of Vγ4Vδ1 TCR reporter cells. In the presence of chimeric anti-BTNL8 mAb or corresponding isotype control, NF-AT-GFP reporter ( A ), CD69 ( B ) and TCRVδ1 ( C ) expression at the plasma membrane of JRT3-Vγ4Vδ1 TCR reporter cells co-cultured with HEK-BTNL3/BTNL8. The dose response of this inhibition as shown by _IC50 / _EC50 assessed for each mAb and each readout.

Figure 5. Anti- BTNL8 mAb inhibits activation of primary Vγ2/3/4 cells. A. CD25 in Vγ2/3/4+Vδ1+ and Vγ2/3/4-Vδ1 assessed by FACS when Vδ1 T cells expanded from HD PBMC were co-cultured with HEK-pLV-null or HEK-BTNL3/BTNL8 Expression on +T cells. B. Effect of anti-BTNL8 mAb on Vγ2/3/4+Vδ1+ cell activation when co-cultured with HEK-BTNL3/BTNL8.

Example: Materials and Methods

Monoclonal Antibody (mAb) Generation Mouse anti-human BTNL8 antibodies were generated by immunizing 48 mice bearing 6 different MHC combinations with recombinant human BTNL8-Fc fusion protein. Mice were bled after 21 days and serum titers of BTNL8-specific polyclonal antibodies were determined by Luminex assay. Mice showing the highest BTNL8-specific antibody titers were euthanized. Splenic B cells were isolated via positive selection and myeloma cells were subjected to PEG-induced fusion for fusion tumor production. Fusions were sub-colonized by limiting dilution, and fusion tumor supernatants underwent two rounds of screening for target specificity and its ability to modulate V[ delta ]i T cell function in vitro. Fusion tumor supernatants were used as is, or mAbs were purified by Protein A resin (GE Healthcare) using an AKTA-Pure apparatus (GE Healthcare).

Luminex Assay Magnetic COOH beads (Biorad) were bound to rhBTNL8-Fc protein (R&D) according to the manufacturer's instructions and the beads were stored in storage buffer (Biorad) at -20°C until use. For titration of mouse serum, serial serum dilutions were performed in Luminex assay buffer (Nanotools) starting at 1:50 with dilution steps of 1:4; 100 μL of bead suspension was mixed with 100 μL of serum dilution, and After incubation for 1 hour at RT, beads were washed 3 times in wash buffer, incubated with 1 μg/mL biotinylated goat anti-mouse IgG-Fc in Luminex assay buffer, and analyzed in Luminex assay. 3 additional washes in buffer. Finally, beads were incubated with 1 μg/mL streptavidin-PE in Luminex assay buffer for 1 hour before 3 final washes in Luminex reading buffer (Nanotools). The beads were resuspended in Luminex reading buffer and data were acquired on a Luminex 100/200 system. For hit identification and affinity assessment, 30 μL of supernatant was transferred to a 96-well plate and 90 μL of Luminex assay buffer was added. One hundred microliters of the bead suspension was mixed with 100 microliters of the supernatant dilution and incubated at RT for 16 hours before proceeding with the protocol described above. For hit identification, those with the highest affinity for the target and the lowest affinity for the irrelevant control protein (Rank-Fc) were selected. FAB beads allow estimation of antibody concentration; the midpoint of its binding curve corresponds to a 100 pM antibody concentration. For affinity/Kd calculations, fusion tumor supernatants were subjected to serial dilutions in Luminex assay buffer starting at 40.000 pM, with dilution steps of 1:4, and assayed as described above. Kd corresponds to the midpoint of the corresponding binding curve. The ratio FAB (MFI)/target (MFI) allows prediction of antibody affinity.

Cell Culture Peripheral blood mononuclear cells (PBMC) were obtained from the leukocyte layer of healthy donors (HD) provided by a local blood bank ( Etablissement Français du Sang (EFS)-Marseille-France), and were obtained by density gradient centrifugation ( Eurobio) separation. The following human cell lines were obtained from the American Type Culture Collection: HEK-293T (embryonic kidney), JRT3 T3.5 cells (Jurkat derivative without αβTCR), HL-60 (myeloid leukemia) cells . HEK-293T cells and derivatives thereof were cultured in DMEM medium (Life Technologies) with 10% fetal calf serum (FCS). HL-60 cells as well as JRT3.3 cells and derivatives thereof were cultured in RPMI 1640 medium supplemented with 10% FCS, 1% sodium pyruvate, 1% L-glutamic acid (all from Life technologies). Fusions were incubated in DMEM/Ham's F12 (1:1) (ThermoFisher Scientific), 4% FetalClone I (Hyclone), Chemically Defined Lipid Concentrate (1:250), Cultured in 1% glutamic acid, 1% sodium pyruvate, and 100 µg/mL PenStrep (all from ThermoFisher Scientific). To collect the fusion tumor supernatant, the fusion tumors were cultured in the absence of Fetalclone for 4 to 5 days.

Lentiviral Transduction To generate lentiviral particles, the following plasmids pMD2.G (encoding the envelope glycoprotein VSV-G), psPAX2 (encoding the HIV-1 derived protein gag ) were used according to the manufacturer's instructions using the TurboFect reagent (ThermoFisher) , pol , tat and Rev ) and the pLV lentiviral expression vector (either empty or encoding the relevant genes as indicated) were transfected into HEK-293T cells. Transfection medium was changed to fresh OptiMEM ^™ (ThermoFisher) after 24 h. Culture supernatants containing lentiviral particles were collected after 24 h and 48 h and concentrated LentiX concentrator™ (Takara) was used following the manufacturer's instructions. For HEK-BTNL8 and HEK-BTNL8/BTNL3 transconductors, wild-type human BTNL8 cDNA (NCBI reference sequence: NM_NM_001040462.2) or with a c-Myc (EQKLISEEDL)-tag in the N-terminus using HindIII/BamHI restriction sites An optimized version of the wild-type human BTNL3 cDNA (NCBI reference sequence: NM_197975.2) was cloned into the pLV vector. HEK-293T cells ( ^2.5 x 105 cells/well) were seeded in a 12-well dish, and 25 µl of concentrated lentiviral particles were added to the culture. After 24 h, cells were washed twice in complete medium and placed back in their regular medium for 48 h. Transducers were selected by adding 1 µg/mL puromycin to the medium.

ELISA analysis of cynomolgus monkey BTNL8 orthologous cross-reactivity After a BLAST search using the human BTNL8 amino acid sequence, the cynomolgus monkey BTNL8 orthologous sequence (XP_005558887.1) was identified and coded using EcoRI/EcoRV restriction sites The ectodomain was cloned into the pFUSE-hlgG1FC2 vector (InvivoGen). Recombinant cynoBTNL8-Fc fusion proteins were generated by transfecting the resulting pFUSE-hlIgG1FC2-cynoBTNL8 plastids into Expi293F ^™ cells with ExpiFectamine™ 293 (ThermoFisher) according to the manufacturer's instructions. Cell culture supernatants collected on day 6 were used for affinity purification column purification. The purified cynoBTNL8-Fc protein was analyzed by SDS-PAGE and Western blotting to measure molecular weight and purity. The cynoBTNL8-Fc protein concentration was determined by Bradford assay using BSA as standard. For ELISA, cynomolgus monkey BTNL8-Fc protein (1 μg/mL in 1×PBS) was coated overnight at 4°C. After 3 washes in PBS, the disks were saturated with 2% v/v BSA in PBS for 1 h at room temperature, and the saturation buffer was discarded. Anti-BTNL8 fusion tumor supernatants were taken ½ dilution in PBS BSA 2% and added 100 µL per well and incubated for 90 minutes at room temperature on a plate shaker. All wells were washed 3 times in PBS before addition of goat anti-mouse IgG HRP (Jackson ImmunoResearch, diluted 1:10000 in PBS BSA 2%) and incubated for 1 h at room temperature. Next, all wells were washed 3 times in PBS and 1-Step ABTS solution (ThermoFisher) was added to show binding as analyzed by absorbance at 405 nm in a Spark luminometer (Tecan).

Generation of Vγ4Vδ1 TCR reporter cells A bicistronic construct containing the human Vγ4 TCR chain and the human Vδ1 TCR chain isolated from the P2A sequence (IC202-g4_P2A_d1: [SEQ ID NO:84]) was designed, synthesized and restricted using BamHI/SalI The loci were cloned into the pLV vector. The NFAT-EF1α-eGFP reporter lentiviral vector was generated using the pLV-Blasticidin-NFAT-EF1α-eGFP vector (custom DNA construct). JRT3 T3.5 cells were simultaneously transduced with lentiviral particles encoding the NFAT-EF1α-eGFP reporter and the IC202-g4_P2A_d1 construct or empty pLV as described above. JRT3 T3.5 transductants were selected in medium containing puromycin (1 µg/mL) and blasticidin (10 µg/mL). The p24 negative status of the transductants was confirmed using the Lenti X ^™ p24 Rapid Titration Kit (Takara). The highest plasma membrane-expressing JRT3 transductors of the Vγ4Vδ1 TCR were sorted by flow cytometry using anti-Vδ1-PE-Vio770 (Miltenyi) in a BD-Aria II cell sorter (Becton Dickinson). In all experiments, JRT3 T3.5 transductants carrying the Vγ4Vδ1 TCR were named JRT3-Vg4Vd1-202 reporter cells.

Analysis of Vγ4Vδ1 TCR reporter cells JRT3-Vg4Vd1-202 reporter cells were combined with HEK-BTNL8/BTNL3 cells (ratio 1:1, 5× each in the presence or absence of 50 µL of anti-BTNL8 or fusion tumor medium (HCM) 10 ⁴ cells; culture volume 100 µL) were co-cultured and incubated at 37°C 5% CO ₂ for 16 hours. Fusion tumor medium (HCM) was added to the co-cultures as a negative control (reference). JRT3-Vg4Vd1-202 reporter cells alone ( ⁵ x 104 cells) were mixed with PMA (20 ng/mL)/ionomycin (1 µg/mL) (Sigma) or anti-CD3 (pure line OKT3) as a positive control ); or incubated with IgG2b, the isotype control for OKT3. At the end of the incubation time, the cells were briefly centrifuged and the pellets were resuspended in a solution containing anti-CD69-APC (BD Biosciences), anti-Vδ1-PE-Vio770 (Miltenyi) and Live/Dead Aqua fluorescent dye (Life Technologies). in the dye mixture. Median fluorescence intensity (MFI) was assessed on an Intellycyt I-QUE cytometer and analyzed with FlowJo software. For each sample, CD69 and TCRVδ1 expression (MFI) at the plasma membrane and the change (Δ%) of the NFAT-eGFP reporter (% of GFP+ cells) from reference were calculated. Anti-BTNL8 antagonists of Vγ4Vδ1 activation were those with the lowest CD69 and NF-AT Δ% below the corresponding median Δ% of all samples and the highest TCRVδ1 Δ% above the median Δ% of all samples.

Expansion of Vδ1 T cells Panγδ T cells were isolated from fresh PBMCs by negative selection using the EasySep ^™ Human γ/δ T Cell Isolation Kit (Stemcell Technologies) following the manufacturer's instructions. Purified γδ T cell lines were grown in mitomycin C _- treated autologous PBMC, Cultured in the presence of human interleukins (rhIL-4, rhIL-1β, rhIL-21 and rhIFN-γ, all from Miltenyi) and soluble anti-CD3 (pure line OKT3, Thermofisher). On day 0, mitomycin C-treated autologous PBMCs were added to the culture as feeder cells (ratio 1:1) and renewed after one week. Every 4 to 5 days, the medium was changed to fresh medium supplemented with interleukins and anti-CD3 mAbs. After one week, rhIL-4 was switched to rhIL-15. The frequency of Vδ1 T cells in the expanded γδ T cells was monitored by flow cytometry on day 0 and weekly during 3 weeks. The expanded Vδ1 T cells were then frozen at -150°C in fetal bovine serum (FBS) supplemented with 10% dimethylsulfoxide (DMSO) for further analysis.

Degranulation analysis of Vδ1 - T cells Cryo-expanded Vδ1 T cells from healthy donors were pre-incubated overnight with human interleukins (rhIL-2 100 UI/mL and rhIL-15 10 ng/mL). The next day, the expanded Vδ1 T cells were co-cultured with a human leukemia cell line expressing BTNL8 (HL-60) as target cells at a 1:1 ratio for 4 hours in the presence of anti-CD107a and anti-CD107b antibodies and Golgistop. During these 4 hour incubations, fifty microliters of anti-BTNL8 fusion tumor supernatant or control medium were added to the co-cultures. Anti-Vδ1 TCR antibody was used as a positive control for Vδ1 T cell activation. After 4 hours of incubation, cells were extracellularly stained with a mixture of fluorescent dye-labeled mAbs (anti-CD3, anti-Panγδ TCR, anti-Vδ1 TCR, and Live/dead) and analyzed by flow cytometry (Cytoflex LX). , Beckman Coulter) analysis. Degranulation was determined by the percentage of CD107ab in Vδ1 T cells (defined as CD3+ Panγδ TCR+Vδ1+).

Flow cytometry PBMC, purified Vδ 1-T cells were quantified prior to analysis using FlowJo 10.5.3 software (FlowJo) on a CytoFlex LX or CytoFlex S (Beckman Coulter) or I-QUE (Sartorius) cytometer Or cell lines were incubated with the indicated mAbs. Antibodies used for Vδ1-T cell degranulation assays were anti- CD107a -FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-Alexa Fluor 700 (Biolegend), anti-PanγδTCR-PE (Miltenyi), Anti- Vδ1TCR PE-Vio770 (Miltenyi) and Live/dead near-infrared IR (Thermofisher). All immunostaining was performed using purified mAb at 10 µg/mL in the presence of FcR blocking reagent (Miltenyi), goat anti-mouse-PE 1:100 (Jackson Immunoresearch) and live/dead near-infrared IR (Thermofisher). Biotinylated anti-myc mAb (ThermoFisher) was used to detect myc-tagged BTNL3 in HEK-BTNL3 cells.

Statistics Results are expressed as median ± SEM for Vδ1 -T cell degranulation. All analyses were performed using GraphPad Prism 7.04 software (GraphPad).

Generation of recombinant chimeric anti -BTNL8 mAbs The VH and VL sequences of reference anti-BTNL8 mAbs were synthesized in vitro and amplified by PCR using PrimeSTAR Max DNA polymerase (Takara). PCR products were cloned into heavy and kappa light chain expression vectors (MI-mAbs) using the In fusion system (Clontech), and plastids were transformed into stem cell competent cells (Clontech). Vector sequencing (MWG Eurofins) was performed in order to verify the parental anti-BTNL8 sequence prior to large-scale (maximum) preparation of plastids for further transfection. Vectors encoding the matched light and heavy chains of each anti-BTNL8 clone were transiently transfected in HEK293-Expi cells (Thermofisher) (2.9 x ¹⁰ cells/ml) at a heavy chain/light chain ratio of 1:1.2 , and the medium was renewed after 18 h. Seven days after transfection, culture supernatants were collected for mAb purification. Affinity purification of antibodies with Protein A Sepharose Fast Flow (GE Healthcare) was performed overnight at 4°C. The binding buffer was 0.5 M glycine, 3 M NaCl, pH 8.9. Elution was performed with the following buffer: 0.1 M citrate pH3. Samples were neutralized immediately after elution with 1M Tris-HCl, pH 9 (10% v/v). Finally, the chimeric anti-BTNL8 mAb was dialyzed into PBS 1× and filtered through a 0.22 μM filter (Millex GV hydrophilic PVDF, Millipore). The chimeric anti-BTNL8 mAb concentration was determined in a Nanodrop 2000 spectrophotometer (ThermoScientific) taking into account the extinction coefficient of the antibody. Purity (as defined by the fraction of mAb monomers) was determined by UPLC-SEC using Acquity UPLC-HClass Bio (Waters) and Acquity UPLC Protein-BEH-200A, 1.7 μm 4.6×50 mm column (Waters). Antibody mass was determined in a Xevo G2-S QT in a mass spectrophotometer (Waters) using a reverse phase column (PLRP-S 4000 A, 5 μm, 50×2.1 mm (Agilent technologies)).

BTNL8 mAb expression on whole blood, epithelial cells and HEK cells Fresh normal adjacent tissues from colorectal adenocarcinoma patients were obtained via BioIVT, and epithelial and immune cells were isolated from these tissues by mechanical dissociation as previously described (Mayassi et al Cell 2019). Heparinized whole blood from healthy donors was obtained from a local blood bank (EFS). The CaCo-2 (colon adenocarcinoma) cell line was purchased from ECACC Collection and cultured in DMEM + 20% FBS + 1 mM NaPy.

Whole blood was performed using purified mAbs (mouse or chimeric mAb) in the presence of FcR blocking reagents (Miltenyi), goat anti-mouse or anti-human PE 1:100 (Jackson Immunoresearch) and live/dead (Thermofisher) , Staining of normal epithelial cells, CaCo-2 and HEK cells. For whole blood, a mixture of fluorescent dye-labeled mAbs (anti-CD15, anti-CD3, anti-CD56, anti-CD19, and anti-CD14) was used to identify neutrophils, T cells, NK cells, B cells and monocytes, and anti-Epcam (Biolegend) mAb was used to identify epithelial cells in tissue-separated cells. For whole blood staining, red blood cells were depleted prior to acquisition using Cal-Lyse Lysis Solution (Thermofisher). Analysis was performed using FlowJo software on a CytoFlex LX or CytoFlex S (Beckman Coulter) or I-QUE (Sartorius) cytometer.

Co-culture of primary Vδ1 T cells with HEK cells Fresh or frozen Vδ1 T cells expanded from healthy donor PBMCs were pre-incubated overnight with rhIL-2 (50 UI/mL) and seeded with HEK cells (pLV-null or BTNL3/ BTNL8) to form a monolayer. The following day, expanded Vδ1 T cells were co-cultured with HEK-pLV-null or HEK-BTNL8 at a 1:1 ratio for 24 hours in the presence of 10 µg/mL anti-BTNL8 mAb or the corresponding isotype control. At the end of the incubation period, cells were treated with fluorescent dye-labeled mAbs (anti-CD3, anti-Panγδ TCR, anti-Vδ1 TCR and anti-Vγ2/3/4 TCR (kindly provided by Pr. Dieter Kabelitz), anti-CD25 and live/dead ) were extracellularly stained and analyzed by flow cytometry (Cytoflex LX, Beckman Coulter).

其中Y=結合水準，Y₀ =締合起始時之結合，A為漸近線且t=時間。k_obs 為所觀測之速率常數。 Binding Affinities by Biolayer Interferometry Analysis Biolayer Interferometry (BLI) was performed as follows: first anti-human IgG Fc was captured in a biosensor (AHC; Fortebio) with 0.2 ml kinetic buffer (PBS pH 7.4, 0.02% Tween20 and 0.1% BSA) were hydrated for 10 min and then loaded with hBTNL8 (2 μg/mL). Association of antibodies to different concentrations of hBTNL8 (40, 20, 10, 5, 2.5 and 1.25 nM) was monitored for 150 s, and dissociation was followed in KB typically for 300 s. All runs (including loading, equilibration, associating/impregnating sensor into analyte, dissociation and regeneration) were performed in black 96-well plates (Greiner) at 30°C with shaking at 1000 rpm using OctetRed96 (ForteBio). A control with an irrelevant antibody is used to assess the absence of nonspecific binding of the target protein. Data fitting and constant measurements were performed with the Octet Red system software (version 11.1) using a 1:1 model. The equation used to fit the association is the integral of the differential equation showing the rate of association as a function of the decreasing concentration of unbound ligand molecules at the onset of analyte binding:

where Y = level of binding, Y ₀ = binding at onset of association, A is the asymptote and t = time. k _obs is the observed rate constant.

其中Y₀ 為在解離開始時之結合，且k_d 為解離速率常數。The equation used to fit the dissociation is:

where Y ₀ is the binding at the onset of dissociation and k _d is the dissociation rate constant.

By solving the above equations for k _obs and k _d , the association rate constant _ka can then be calculated by the following equation:

Finally, use _ka and k _d to calculate the affinity constant K _D :

Epitope Binning by Biolayer Interferometry Analysis Epitope binning was performed using BLI as described above. The test antibody is immobilized on the sensor. Recombinant human BTNL8 was then present at 40 nM, and when the association reached saturation, the second (competing) antibody was present at 40 nM. If the competing antibody and the test antibody bind overlapping epitopes, no additional binding curves will be observed. A second binding curve will be observed if the competing antibody binds to a non-overlapping epitope. Test antibodies are present in free ligand form to demonstrate lack of binding to overlapping epitopes and use this signal as a reference. Grouped data were analyzed using the Epitope Bin operation using Octet Data Analysis HT 11.1.

Results Reference anti- BTNL8 fusion tumors were identified as follows. Reference anti-BTNL8 antibodies were identified as follows: Mice were immunized with BTNL8-Fc antigen from mice, and splenocytes presenting the highest titers of BTNL8-specific serum were collected and combined with Myeloma is fused to obtain a fusion tumor. Fusion tumor culture supernatants showing the highest affinity for BTNL8 (n=320), as assessed on the Luminex assay, underwent a first round of screening based on their ability to bind human BTNL8 intracellularly. As shown in Figure 1C, 310/320 anti-BTNL8 fusion tumor supernatants were able to stain HEK-BTNL8, and all supernatants stained HEK-BTNL8/BTNL3 transductants but not wild-type HEK293T cells or HEK-BTNL3 transductants, As assessed on flow cytometry. To identify clones that exhibit immunomodulatory properties on Vδ1 T cells, anti-BTNL8 fusion tumor supernatants were tested for their ability to modulate Vγ4Vδ1 TCR reporter cell activation and subsequently the cytotoxicity of expanded Vδ1 T cells against BTNL8-expressing cancer cells. The clones selected from this first round of screening were subjected to secondary colonization, which resulted in 81 secondary clones retested against the same criteria against BTNL8 fusionomas.

BTNL8 targeting with anti- BTNL8 mAb allows modulation of BTNL8/BTNL3 -induced activation via the Vγ4Vδ1 TCR. JRT3-T3.5 T cells, a derivative of the Jurkat T cell line without TCRαβ, were transduced to express the colon-derived Vγ4Vδ1 TCR and GFP reporter transgenes under the control of the NF-AT promoter. These cells are referred to as JRT3-Vg4Vd1-202 reporter cells. At the same time, HEK-293 cells stably expressing BTNL8 and BTNL3 (HEK-L8/L3) were generated. Given the ability of the BTNL8/BTNL3 dimer to trigger activation via the Vγ4Vδ1 TCR in intestinal Vδ1 T cells (Di Marco Barros et al. Cell, 2016), we evaluated anti-BTNL8 fusion tumor supernatants compared to unrelated fusion tumor supernatants Whether the solution could modulate the activation induced by co-culture of JRT3-Vg4Vd1-202 reporter cells with HEK-BTNL8/BTNL3 cells (Figure 2A and B). It was found that forty-seven anti-BTNL8 fusion tumor supernatants regulated BTNL8/BTNL3-induced JRT3-Vg4Vd1-induced BTNL8/BTNL3-induced JRT3-Vg4Vd1- 202 reporter activation (Figure 2B). Specifically, thirty-five anti-BTNL8 fusion tumor supernatants induced up-regulation of TCRVδ1 expression and down-regulation of NFAT-GFP reporter and CD69 (Figure 2C and Table 3). Forty-seven clones including these 35 anti-BTNL8 fusion tumor supernatants were selected for the next step of screening and subselection. Table 3 : Anti- BTNL8 fusion tumor supernatants selected on the BTNL8/BTNL3-JRT3-Vg4Vd1-202 reporter assay . Changes in NFAT-GFP reporter, CD69 and TCRVδ1 expression (Δ) compared to co-cultures of JRT3-Vg4Vd1-202 reporter cells with HEK-BTNL8/BTNL3 in the presence of fusion tumor medium. fusion tumor fusion Fusion tumor ID TCRVd1 (MFI) CD69(MFI) NFAT+ cells (%) TCR Vd1 (%*) TCRVd1 (Δ%) CD69 (%*) CD69 (Δ%) NF-AT (%*) NF-AT (Δ%) B MP1A08 6249 3171 2.45 222.5 122.5 40.8 -59.2 18.4 -81.6 A MP1H03 3539 5080 5.93 126.0 26.0 65.3 -34.7 44.6 -55.4 A MP1H05 6224 2819 2.59 221.7 121.7 36.2 -63.8 19.5 -80.5 B MP1F06 6723 3184 4.87 239.4 139.4 40.9 -59.1 36.6 -63.4 B MP1C07 6534 2808 1.2 232.7 132.7 36.1 -63.9 9.0 -91.0 B MP1B09 5808 2924 1.87 206.8 106.8 37.6 -62.4 14.1 -85.9 B MP1A10 5808 3120 5.98 206.8 106.8 40.1 -59.9 45.0 -55.0 B MP2B3 6879 3229 3.27 294.6 194.6 61.1 -38.9 32.1 -67.9 B MP2C4 4372 5178 9.84 187.2 87.2 97.9 -2.1 96.5 -3.5 B MP2A6 7107 2739 1.32 304.4 204.4 51.8 -48.2 12.9 -87.1 B MP2A7 7159 2423 0.28 306.6 206.6 45.8 -54.2 2.7 -97.3 B MP2E7 4349 4139 7.39 186.3 86.3 78.3 -21.7 72.5 -27.5 B MP2C8 5749 3833 4.64 246.2 146.2 72.5 -27.5 45.5 -54.5 B MP2A8 7953 2617 0.71 340.6 240.6 49.5 -50.5 7.0 -93.0 B MP2F8 6317 2466 0.57 270.5 170.5 46.6 -53.4 5.6 -94.4 B MP2B9 5907 3969 6.6 253.0 153.0 75.1 -24.9 64.7 -35.3 B MP2C9 6114 2432 0.39 261.8 161.8 46.0 -54.0 3.8 -96.2 B MP2G9 6622 2730 1.36 283.6 183.6 51.6 -48.4 13.3 -86.7 B MP3F1 4369 4456 6.05 161.0 61.0 62.2 -37.8 55.0 -45.0 B MP3A2 4363 4115 5.64 160.8 60.8 57.4 -42.6 51.3 -48.7 B MP3B3 6720 2800 0.5 247.6 147.6 39.1 -60.9 4.5 -95.5 B MP3A5 6771 2511 0.51 249.5 149.5 35.0 -65.0 4.6 -95.4 B MP3E5 7003 2785 0.98 258.0 158.0 38.8 -61.2 8.9 -91.1 B MP3F7 6993 2504 0.48 257.7 157.7 34.9 -65.1 4.4 -95.6 B MP4C09 6274 3568 4.4 235.6 135.6 53.7 -46.3 46.1 -53.9 B MP4B01 6249 2696 0.84 234.7 134.7 40.6 -59.4 8.8 -91.2 B MP4A04 6534 2320 0.37 245.4 145.4 34.9 -65.1 3.9 -96.1 B MP4C06 6224 3236 3.08 233.7 133.7 48.7 -51.3 32.3 -67.7 B MP4D06 4998 3981 4.12 187.7 87.7 59.9 -40.1 43.2 -56.8 B MP4H05 6945 2329 0.28 260.8 160.8 35.1 -64.9 2.9 -97.1 B MP4E07 7321 2578 0.37 274.9 174.9 38.8 -61.2 3.9 -96.1 B MP4D08 6455 2842 1.79 242.4 142.4 42.8 -57.2 18.8 -81.2 B MP4H08 6641 2406 0.29 249.4 149.4 36.2 -63.8 3.0 -97.0 B MP4G09 6973 2455 0.41 261.8 161.8 37.0 -63.0 4.3 -95.7 B MP4H10 5142 3468 3.29 193.1 93.1 52.2 -47.8 34.5 -65.5

Anti- BTNL8 antibodies induce degranulation of Vδ1 T cells against BTNL8 - expressing cancer cells Although BTNL8/BTNL3-mediated activation of the Vγ4Vδ1 TCR has been described in the literature (Melandri et al. Nat Immunol, 2018), it is not effective in Vδ1 T cells. The final result is still unknown. We hypothesized that BTNL8/BTNL3-mediated Vγ4Vδ1 TCR activation may be involved in Vδ1 T cell cytotoxicity. To test this hypothesis, purified Vδ1 T cells were expanded from PBMCs of healthy donors and co-cultured with HL-60 leukemia cells in the presence or absence of anti-BTNL8 fusion supernatants. BTNL8 was expressed at the plasma membrane in the case of (Figure 3A) as target cells. Interestingly, we found that expanded Vδ1 T cell degranulation against HL-60 leukemia cells was enhanced 2- to 3-fold by anti-Vδ1 TCR antibodies (FIG. 3B). More importantly, 23 of the 47 anti-BTNL8 fusion tumor supernatants initially selected for activity in the regulatory reporter assay regulated Vδ1 T cell degranulation. Indeed, the addition of 23 anti- BTNL8 fusion tumor supernatants resulted in increased lysis of expanded Vδ1 T cells compared to co-cultures with HL-60 cells alone or in the presence of control fusion tumor medium Inhibition, as measured by the percentage of CD107+ degranulated cells. As expected, PMA/ionomycin treatment of V[ delta ]i T cells resulted in maximal induction of their lytic function without addition of HL-60 cells.

The percentage of CD107+ cells induced by anti-BTNL8 fusion tumor supernatant ranged from 7.98% to 46.4% CD107+ cells compared to co-cultures with control fusion tumor medium (17.2%). In co-cultures of expanded V δ 1 T cells with BTNL8 expressing HL-60 cells, anti-BTNL8 supernatants elicited expanded V compared to the same co-cultures in the presence of control fusion tumor medium 1.44- to 2-fold inhibition of delta 1 T cell degranulation.

To confirm the Vδ1 T cell antagonist activity found against some of the anti-BTNL8 fusion tumor supernatants during primary screening, primary hits were sub-clone and anti-BTNL8 mAbs were purified from the resulting sub-colonized fusion tumor supernatants. Purified anti-BTNL8 fusion tumor supernatants were screened for their ability to modulate Vδ1 T cell degranulation against HL-60 cells. Twenty-four purified anti-BTNL8 mAbs were found to reduce the inhibition of degranulation of Vδ1 T cells against HL-60 cells by ≥15% compared to their control isotype mAbs. Table 4 shows the percentage of degranulated (CD107ab+) Vδ1 cells obtained in the presence of these 24 antagonist anti-BTNL8 mAbs tested against 3 different healthy donors. Table 4 : Vδ1 T cell degranulation against HL-60 cells in the presence of Vδ1 antagonist anti- BTNL8 mAb pure line CD107 Vd1 + T cells % % CD107 Vd1+ T cells : normalized data Donor A Donor B Donor C Donor A Donor B Donor C median mIgG1 11.30 12.90 17.30 1.00 1.00 1.00 1.00 mIgG2b 11.90 13.60 20.00 1.00 1.00 1.00 1.00 1 mAb1 6.59 9.62 12.80 0.58 0.75 0.74 0.74 2 mAb2 7.46 11.00 14.40 0.66 0.85 0.83 0.83 3 mAb3 (mAbX1) 8.96 10.60 13.50 0.79 0.82 0.78 0.79 4 mAb4 9.72 10.00 13.60 0.86 0.78 0.79 0.79 5 mAb5 (mAbX3) 9.29 10.90 13.00 0.82 0.84 0.75 0.82 6 mAb6 (mABX2) 10.90 9.83 13.40 0.96 0.76 0.77 0.77 7 mAb7 7.14 9.21 13.50 0.63 0.71 0.78 0.71 8 mAb9 6.53 8.74 12.90 0.58 0.68 0.75 0.68 9 mAb10 7.00 9.12 12.80 0.62 0.71 0.74 0.71 10 mAb14 6.41 9.55 12.10 0.57 0.74 0.70 0.70 11 mAb15 6.79 8.52 12.60 0.60 0.66 0.73 0.66 12 mAb16 10.00 10.70 14.50 0.88 0.83 0.84 0.84 13 mAb17 9.04 7.96 13.40 0.80 0.62 0.77 0.77 14 mAb19 (mAbX5) 9.54 10.00 13.80 0.84 0.78 0.80 0.80 15 mAb20 7.19 10.40 13.10 0.64 0.81 0.76 0.76 16 mAb21 7.93 9.10 12.40 0.70 0.71 0.72 0.71 17 mAb26 6.82 9.57 12.80 0.60 0.74 0.74 0.74 18 mAb29 (mAbX4) 7.52 11.30 13.40 0.67 0.88 0.77 0.77 19 mAb34 7.44 8.32 11.70 0.66 0.64 0.68 0.66 20 mAb38 9.20 10.80 14.10 0.81 0.84 0.82 0.82 twenty one mAb40 6.57 9.34 13.00 0.58 0.72 0.75 0.72 twenty two mAb41 8.96 9.92 15.30 0.79 0.77 0.88 0.79 twenty three mAb42 8.45 9.65 13.00 0.75 0.75 0.75 0.75 twenty four mAb47 10.40 10.40 15.00 0.87 0.76 0.75 0.76 Vd1 + PMA/ ionomycin 87.30 93.30 87.00 culture medium 12.20 12.50 14.80

Reference Vδ1 antagonist anti- BTNL8 mAb recognizes intracellular BTNL8 but not BTNL3 To confirm that the identified Vδ1 antagonist anti-BTNL8 antibodies recognize BTNL8 but not its molecular partner BTNL3, lentiviral vectors encoding BTNL8 or BTNL3 or both were used Transduction of HEK-293T cells. In all clones except mAb34 and mAb47, the selected 24 anti-BTNL8 mAbs were only detected in HEK-BTNL8 and HEK-BTNL8/BTNL3 cells alone, but not in HEK-293T WT or HEK-BTNL3 cells. Staining of 293T WT, HEK-BTNL8, HEK-BTNL3 or HEL-BTNL8/BTNL3 (data not shown).

mAb34 showed weaker staining of HEK-293T WT and HEK-BTNL3. mAb47 showed staining for HEK-BTNL3, indicating cross-reactivity with BTNL3.

After sequencing these anti-BTNL8 mAbs, we provide the sequences of 5 reference Vγ1 antagonist antibodies that display Vγ1 antagonist activity and bind to HEK-BTNL8, HEK BTNL8/BTNL3 but not HEK in Table 10 -BTNL3.

Affinity of reference Vδ1 antagonist anti- BTNL8 antibody mAb for BTNL8 The affinity of reference anti-BTNL8 antibody for recombinant human BTNL8-Fc protein was assessed by testing its corresponding fusion tumor supernatant using Luminex. The data are summarized in Table 5 . Table 5 : Luminex -based assessment of affinity of reference anti- BTNL8 mAbs pure line isotype K _D (pM) BTNL8-Fc mAb3 (mAbX1) IgG1 <10 mAb5 (mAbX3) IgG1 <10 mAb6 (mAbX2) IgG1 <10 mAb19 (mAb X5) IgG1 <10 mAb29 (mAbX4) IgG1 <10

Cross-reactivity of reference anti- BTNL8 mAb to cynomolgus BTNL8 orthologs BTNL8 orthologs are present in most non-human primates, including cynomolgus monkeys ( Macaca fascicularis ). To determine the cross-reactivity of the reference anti-BTNL8 mAb with the cynomolgus monkey BTNL8 ortholog (cynoBTNL8; NCBI reference XP_005558887.1, 88% identical to human BTNL8), we generated recombinant Fc fusions containing the extracellular domain of cynoBTNL8 protein (cynoBTNL8-Fc), and we performed an ELISA assay for evaluating the binding of fusion tumor supernatants from the reference anti-BTNL8 mAb to this protein. As shown in Table 6, the reference Vδ1 antagonistic anti-BTNL8 mAb was able to bind cynoBTNL8-Fc (optical density (OD) at 405 nm) > 0.2 in our ELISA assay compared to an irrelevant control mAb (anti-His). The set threshold value exceeds the absorbance of the fusion tumor medium (HCM). Three of the 18 Vδ1 antagonistic anti-BTNL8 mAbs (mAb1, mAb9, mAb34) did not bind to cynoBTNL8-Fc, implying no cross-reactivity with the cynomolgus BTNL8 ortholog (data not shown). As expected, the anti-Fc mAb used as a positive control for cynoBTNL8-Fc binding produced a strong positive signal (OD _{405 nm} = 1.293). Table 6 : ELISA assessment of binding of reference V [ delta ] 1 antagonistic anti- BTNL8 fusion tumor supernatants to cynomolgus monkey BTNL8 orthologs . pure line OD _{405 nm} mAb3 (mAbX1) 0.696 mAb5 (mAbX3) 0.417 mAb6 (mAbX2) 0.528 mAb19 (mAb X5) 0.553 mAb29 (mAbX4) 0.350 HCM 0.160 Anti-His 0.184 anti-Fc 1.293

Affinity, cross-reactivity and epitope grouping of reference anti- BTNL8 mAbs The affinity of the chimeric version of the reference mAb was first assessed by flow cytometry for its intracellular binding. In cells, all reference anti-BTNL8 mAbs, but not mAbX3, bound BTNL8 and BTNL3/BTNL8 heterodimers on HEK cells with high affinity (< 0.5 µg/ml = 3.3 nM) (data not shown) and presented corresponding EC _50s In Table 7 below: Table 7 : Binding of 4 selected chimeric anti- BTNL8 mAbs to HEK-BTNL8 cells by flow cytometry mAbX1 mAbX2 mAbX4 mAbX5 EC50 (µg/mL) 0.038 0.217 0.343 0.286 EC50 (nM) 0.253 1.449 2.286 1.905

Similar results were obtained for binding to recombinant BTNL8 protein as determined by biolayer interferometry (BLI), and the affinity constants (KD, _kd , _ka ) of selected anti- _BTNL8 chimeric mAbs are presented in the table below: Table 8 : Kinetic parameters of chimeric antibodies binding to human BTNL8 protein as determined by biolayer interferometry (BLI) mAb K _D (M) k _a (1/Ms) K _d (l/s) Full R ² Full Chi ² mAbX1 1.16E-10 3.58E+05 3.84E-05 0.999 0.112 mAbX2 2.52E-10 5.59E+05 1.40E-04 0.999 0.330 mAbX3 unbound mAbX4 9.11E-10 4.54E+05 4.12E-04 0.999 0.274 mAbX5 1.61E-10 5.69E+05 8.71E-05 0.999 0.261

mAbX1, mAbX2, mAbX4 and mAbX5 bound BTNL8 with nanomolar affinity, and mAbX3 was found not to bind BTNL8.

In addition, mAbX1, mAbX2, mAbX4 and mAbX5 bound to recombinant cynoBTNL8-Fc protein, indicating cross-reactivity of the reference anti-BTNL8 mAb with the cynomolgus BTNL8 ortholog (data not shown).

Subsequently, we investigated whether the reference mAb recognized the same BTNL8 epitope region. Therefore, an octet-based grouping experiment was performed in which 4 binder mAbs competed for BTNL8 binding using a "classical sandwich" setup. mAbX1 did not block the binding of the other 3 mAbs to BTNL8, but mAbX2, mAbX4, and mAbX5 blocked the binding of each other, indicating that these mAbs bind to overlapping epitope regions on BTNL8, and mAbX1 binds to different epitopes, as follows Shown in the tables: Table 9 : Pearson correlation coefficient matrix between saturating mAbs ( mAbX1 , rows ) and blocking mAbs ( mAbX2 , columns ) . The strongest correlations are shown in black, and the weaker correlations are shown in white. Antibody self-binding pair values are presented in bold. mAbX2 mAbX5 mAbX4 mAbX1 mAbX2 0.108 0.107 0.124 0.293 mAbX5 0.091 0.088 0.103 0.332 mAbX4 0.070 0.080 0.087 0.301 mAbX1 0.349 0.311 0.331 0.121

Reference anti- BTNL8 mAb binds to BTNL8 on normal and tumor epithelial cells from human intestinal tissue ( data not shown ) on intestinal epithelial cells, neutrophils and CaCo-2 cells according to the Human Protein Atlas High levels of BTNL8 mRNA were detected. We determined BTNL8 expression at the plasma membrane of primary normal and tumor intestinal cells by using our reference anti-BTNL8 mAb. mAbX1, mAbX2, mAbX4 and mAbX5 stained Caco-2 cells with variable intensities. Strong staining of primary epithelial cells (Epcam+ cells) from normal tissue was also observed using mAbX1. No binding of these reference anti-BTNL8 mAbs was detected on immune cells from peripheral blood, including neutrophils.

Reference anti- BTNL8 mAb potently inhibits Vγ4Vδ1 T cell activation induced by BTNL3 /BTNL8 dimers . Previous data obtained with fusion tumor supernatants showed that reference BTNL8 mAb acts as an antagonist of Vγ4Vδ1 TCR reporter cell activation as determined by qualitative Demonstrated by increased expression of TCRVδ1 at the membrane and inhibition of NFAT-GFP reporter and CD69 expression. This antagonist activity was also demonstrated when using a reference chimeric anti-BTNL8 mAb (Figure 4). The antagonistic activity of the chimeric anti-BTNL8 mAb was more potent, as demonstrated by _IC50 / _EC50 < 0.5 μg/mL (< 3.3 nM) for all reads tested (Figure 4).

Reference anti- BTNL8 mAb is able to inhibit primary Vγ4Vδ 1 T cell activation induced by BTNL3 /BTNL8 dimer We sought to determine whether reference anti-BTNL8 mAb could modulate primary Vd1 T cell activation. To address this issue, we first tested the ability of BTNL3/BTNL8 dimers to activate primary Vd1 T cell subsets expanded from healthy PBMCs.

Co-culture with HEK-BTNL3/L8 cells induced consistent activation of Vγ2/3/4+ Vδ1 T cells compared to HEK-pLV null cells in all donors tested (Figure 5A). This activation was inhibited by mAbX1, mAbX2, mAbX4 and mAbX5 but not by mAbX3 (Figure 5B). No relevant effect on the Vγ2/3/4-Vδ1 T cell subset was observed (data not shown).

The weaker antagonist activity of the reference anti-BTNL8 mAb in this assay compared to the Vγ4Vδ1 TCR reporter assay may be due to the heterogeneity in the proportion of Vγ4-bearing cells within primary Vγ2/3/4+ Vδ1 T cells from different samples, As detected by anti-Vγ2/3/4 mAb. Table 10 : Brief description of suitable amino acid and nucleotide sequences for use in practicing the invention (highlighting CDR regions in VH/VL sequences) SEQ ID NO: A brief description 1 VH mAb X1 aa sequence QVQLQQSGAELARPGASVKLSCKASGYIFT SYGIG WVKQRTGQGLEWIG EVYPRNGHSYYNEKFKG KATLTADRSSSTAYMQLSSLTSEDSAVYFCARGGTGSQLDYWGQGTTLTVSS 2 VL mAb X1 aa sequence DIQMTQSPASLSASVGETVTITC RASENIYSYLA WYQQKQGKSPHLLVY NAKTLA EGVPSRFSGSGSGTQFSLRISLQPEDFGNYYC QHHYGTYT FGGGTKLEIK 3 VH mAb X2 aa sequence EVQLQQSGAEFVRPGASVKMSCTGSGLNIE DDYMH WVKQRPEQGLEWIG RIDPANGNTKYGPKFQD KATITADTSSNTAYLQLSSLTAEDTAVYYCTR NYYDGSLYYFDY WGQGTTLTVSS 4 VL mAb X2 aa sequence QIVLTQSPAIMSASLGEEITLTC SASSSVSYMH WYQQKSGTSPKLLIY STSNLAS GVPSRFSGSGSGTFYSLTISSVEAEDAADYYC HQWSRYPYT FGGGTKLEIK 5 VH mAb X3 aa sequence EVQLQQSGAELVRPGASVKLSCTASGFNIK DDYMH WVKQRPEQGLEWIG RIDPANANTKYAPKFQD KATITADTSSNTAYLHLSSLTSEDSAVYYCAR NYYDGSPYYFDYWGQGTTLTVSS 6 VL mAb X3 aa sequence NIVMTQSPKSMSMSVGERVTLTC KASENVVTYVS WYQQKPEQSPKLLIY GASNRYT GVPDRFTGSGSATDFTLTISSVQAEDAAGYYC HQWSRYPYT FGGGTKLEIK 7 VH mAb X4 aa sequence EVQLQQSGAELVRPGASVKLSCTASGFNIK DDYMH WVKQRPEQALEWIG RIDPANGNTKYVPKFQD KATITADTSSNTASLQLSSLTSEDTAVYYCAR WGIYDGYYYTMDSWGQGTSVTVSS 8 VL mAb X4 aa sequence QNVLIQSPAIMSASPGEKVTITC SASSSVSYMH WFQQKPGTSPKLWIY STSNLAS GVPARFSGSGSGTSYSLTISRMEAADAATYFC QQRSRYPYT FGGGTKLEIK 9 VH mAb X5 aa sequence EVQLQQSGAELVRPGASIKLSCTASGFNIE DDYMH WVRQRPVQGLEWIG RIDPANGNANFAPKFQD KATITADTSSNTAYLQLSSLASEDTAVYYCTR EGGLYYGNSDAMDYWGQGTSVTVSS 10 VL mAb X5 aa sequence QIVLTQSPAIMSASPGEKVTITC SARSSVSYMQ WIQQKPGTSPKLWIY STSNLAS GVPDRFSGSGSGTSYSLTISRMEAEDAATYYC QQRSRYPRT FGGGTKLEIK 11 mAb X1 HCDR1 aa SYGIG 12 mAb X1 HCDR2 aa EVYPRNGHSYYNEKFKG 13 mAb X1 HCDR3 aa GGTGSQLDY 14 mAb X1 LCDR1 aa RASENIYSYLA 15 mAb X1 LCDR2 aa NAKTLAE 16 mAb X1 LCDR3 aa QHHYGTYT 17 mAb X2 HCDR1 aa DDYMH 18 mAb X2 HCDR2 aa RIDPANNTKYGPKFQD 19 mAb X2 HCDR3 aa NYYDGSLYYFDY 20 mAb X2 LCDR1 aa SASSSVSYMH twenty one mAb X2 LCDR2 aa STSNLAS twenty two mAb X2 LCDR3 aa HQWSRYPYT twenty three mAb X3 HCDR1 aa DDYMH twenty four mAb X3 HCDR2 aa RIDPANANTKYAPKFQD 25 mAb X3 HCDR3 aa NYYDGSPYYFDY 26 mAb X3 LCDR1 aa KASENVVTYVS 27 mAb X3 LCDR2 aa GASNRYT 28 mAb X3 LCDR3 aa HQWSRYPYT 29 mAb X4 HCDR1 aa DDYMH 30 mAb X4 HCDR2 aa RIDPANNTKYVPKFQD 31 mAb X4 HCDR3 aa WGIYDGYYYTMDS 32 mAb X4 LCDR1 aa SASSSVSYMH 33 mAb X4 LCDR2 aa STSNLAS 34 mAb X4 LCDR3 aa QQRSRYPYT 35 mAb X5 HCDR1 aa DDYMH 36 mAb X5 HCDR2 aa RIDPANGNANFAPKFQD 37 mAb X5 HCDR3 aa EGGLYYGNSDAMDY 38 mAb X5 LCDR1 aa SARSSVSYMQ 39 mAb X5 LCDR2 aa STSNLAS 40 mAb X5 LCDR3 aa QQRSRYPRT 41 mAb X1 HCDR1 nt AGCTATGGTATAGGC 42 mAb X1 HCDR2 nt GAGGTTTATCCTAGAAATGGTCATTCTTACTACAATGAGAAGTTCAAGGGC 43 mAb X1 HCDR3nt GGGGGGACGGGAAGCCAACTTGACTAC 44 mAb X1 LCDR1 nt CGAGCAAGTGAGAATATTTACAGTTATTTAGCA 45 mAb X1 LCDR2 nt AATGCAAAAACCTTAGCAGAG 46 mAb X1 LCDR3 nt CAACATCATTATGGTACTTACACG 47 mAb X1 VHnt CAGGTTCAACTGCAGCAGTCTGGAGCTGAGCTGGCGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTATATCTTCACA AGCTATGGTATAGGC TGGGTGAAGCAGAGGACTGGACAGGGCCTTGAGTGGATTGGA GAGGTTTATCCTAGAAATGGTCATTCTTACTACAATGAGAAGTTCAAGGGC AAGGCCACACTGACTGCAGACAGGTCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGA GGGGGGACGGGAAGCCAACTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 48 mAb X1 VL nt GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGT CGAGCAAGTGAGAATATTTACAGTTATTTAGCA TGGTATCAGCAGAAACAGGGAAAATCTCCTCACCTCCTGGTCTAT AATGCAAAAACCTTAGCAGAG GGTGTGCCATCAAGATTCAGTGGCAGTGGCTCCGGCACACAGTTTTCTCTGAGGATCAGCAGCCTGCAACCTGAAGATTTTGGGAATTATTACTGT CAACATCATTATGGTACTTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA 49 mAb X2 HCDR1 nt GACGACTATATGCAC 50 mAb X2 HCDR2 nt AGGATTGATCCTGCGAATGGTAATACTAAATATGGCCCGAAGTTCCAGGAC 51 mAb X2HCDR3nt AATTACTACGATGGTAGTTTGTACTACTTTGACTAC 52 mAb X2 LCDR1 nt AGTGCCAGCTCGAGTGTTAGTTACATGCAC 53 mAb X2 LCDR2 nt AGCACATCCAACCTGGCTTCT 54 mAb X2 LCDR3 nt CATCAGTGGAGTCGTTATCCGTACACG 55 mAb X2 VHnt GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGTTTGTGAGGCCAGGGGCCTCAGTCAAGATGTCCTGTACAGGTTCTGGCTTAAACATTGAA GACGACTATATGCAC TGGGTGAAGCAGCGGCCTGAACAGGGCCTGGAGTGGATTGGA AGGATTGATCCTGCGAATGGTAATACTAAATATGGCCCGAAGTTCCAGGAC AAGGCCACTATAACTGCCGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACAGCTGAGGACACTGCCGTCTATTACTGTACTAGA AATTACTACGATGGTAGTTTGTACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 56 mAb X2VLnt CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCTAACCTGC AGTGCCAGCTCGAGTGTTAGTTACATGCAC TGGTACCAGCAGAAGTCAGGCACTTCTCCCAAACTCTTGATTTAT AGCACATCCAACCTGGCTTCT GGAGTCCCTTCTCGCTTCAGTGGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGCAGTGTGGAGGCTGAAGATGCTGCCGATTATTACTGC CATCAGTGGAGTCGTTATCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA 57 mAb X3 HCDR1nt GACGACTATATGCAC 58 mAb X3 HCDR2nt AGGATTGATCCTGCTAATGCTAATACTAAATATGCCCCGAAGTTCCAGGAC 59 mAb X3 HCDR3 nt AATTACTACGATGGTAGCCCTTACTACTTTGACTAC 60 mAb X3 LCDR1 nt AAGGCCAGTGAGAATGTGGTTACTTATGTTTCC 61 mAb X3 LCDR2 nt GGGGCATCCAACCGGTACACT 62 mAb X3 LCDR3 nt CATCAGTGGAGTCGTTATCCATACACG 63 mAb X3 VHnt GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTAAA GACGACTATATGCAC TGGGTGAAGCAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGA AGGATTGATCCTGCTAATGCTAATACTAAATATGCCCCGAAGTTCCAGGAC AAGGCCACTATAACTGCAGACACATCCTCCAACACAGCCTACCTGCACCTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCTCGA AATTACTACGATGGTAGCCCTTACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 64 mAb X3 VL nt AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGC AAGGCCAGTGAGAATGTGGTTACTTATGTTTCC TGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATAC GGGGCATCCAACCGGTACACT GGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGATGCTGCCGGTTATTACTGC CATCAGTGGAGTCGTTATCCATACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA 65 mAb X4 HCDR1nt GACGACTATATGCAC 66 mAb.X4HCDR2nt AGGATTGATCCTGCGAATGGTAATACTAAAATATGTCCCGAAGTTCCAGGAC 67 mAb X4 HCDR3nt TGGGGAATCTATGACGGTTACTACTATACTATGGACTCC 68 mAb X4 LCDR1 nt AGTGCCAGCTCAAGTGTAAGTTACATGCAC 69 mAb X4 LCDR2nt AGCACATCCAACCTGGCTTCT 70 mAb X4 LCDR3nt CAGCAGAGGAGTAGGTACCCGTACACG 71 mAb X4 VHnt GAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCGGGGGCCTCAGTCAAGTTGTCCTGCACAGCCTCTGGCTTTAACATTAAA GACGACTATATGCAC TGGGTGAAACAGAGGCCTGAACAGGCCCTTGAGTGGATTGGA AGGATTGATCCTGCGAATGGTAATACTAAATATGTCCCGAAGTTCCAGGAC AAGGCCACTATAACTGCAGACACTTCCTCCAACACAGCCTCCCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGA TGGGGAATCTATGACGGTTACTACTATACTATGGACTCC TGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA 72 mAb X4 VL nt CAAAATGTTCTCATCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGC AGTGCCAGCTCAAGTGTAAGTTACATGCAC TGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTAT AGCACATCCAACCTGGCTTCT GGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGCAGATGCTGCCACTTATTTCTGC CAGCAGAGGAGTAGGTACCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA 73 mAb X5 HCDR1nt GACGACTATATGCAC 74 mAb X5 HCDR2nt AGAATTGATCCTGCGAATGGTAATGCTAATTTTGCCCCGAAGTTCCAGGAC 75 mAb X5 HCDR3nt GAGGGAGGCCTCTACTATGGTAACTCGGATGCTATGGACTAC 76 mAb X5 LCDR1 nt AGTGCCAGGTCAAGTGTAAGTTACATGCAG 77 mAb X5 LCDR2nt AGCACATCCAACCTGGCTTCT 78 mAb X5 LCDR3nt CAGCAAAGGAGTCGTTACCCACGGACG 79 mAb X5 VHnt GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTCAATCAAGTTGTCCTGCACAGCTTCTGGCTTTAACATTGAA GACGACTATATGCAC TGGGTGAGGCAGAGGCCTGTTCAGGGCCTGGAGTGGATTGGA AGAATTGATCCTGCGAATGGTAATGCTAATTTTGCCCCGAAGTTCCAGGAC AAGGCCACTATAACTGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGGCATCTGAGGACACTGCCGTCTATTACTGTACTAGA GAGGGAGGCCTCTACTATGGTAACTCGGATGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 80 mAb X5 VL nt CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGC AGTGCCAGGTCAAGTGTAAGTTACATGCAG TGGATCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTAT AGCACATCCAACCTGGCTTCT GGAGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGC CAGCAAAGGAGTCGTTACCCACGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA 81 hBTNL8aa MALMLSLVLSLLKLGSGQWQVFGPDKPVQALVGEDAAFSCFLSPKTNAEAMEVRFFRGQFSSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAEGRISLRLENITVLDAGLYGCRISSQSYYQKAIWELQVSALGSVPLISITGYVDRDIQLLCQSSGWFPRPTAKWKGPQGQDLSTDSRTNRDMHGLFDVEISLTVQENAGSISCSMRHAHLSREVESRVQIGDTFFEPISWHLATKVLGILCCGLFFGIVGLKIFFSKFQWKIQAELDWRRKHGQAELRDARKHAVEVTLDPETAHPKLCVSDLKTVTHRKAPQEVPHSEKRFTRKSVVASQSFQAGKHYWEVDGGHNKRWRVGVCRDDVDRRKEYVTLSPDHGYWVLRLNGEHLYFTLNPRFISVFPRTPPTKIGVFLDYECGTISFFNINDQSLIYTLTCRFEGLLRPYIEYPSYNEQNGTPIVICPVTQESEKEASWQRASAIPETSNSESSSQATTPFLPRGEM 82 hBTNL3 aa MAFVLILVLSFYELVSGQWQVTGPGKFVQALVGEDAVFSCSLFPETSAEAMEVRFFRNQFHAVVHLYRDGEDWESKQMPQYRGRTEFVKDSIAGGRVSLRLKNITPSDIGLYGCWFSSQIYDEEATWELRVAALGSLPLISIVGYVDGGIQLLCLSSGWFPQPTAKWKGPQGQDLSSDSRANADGYSLYDVEISIIVQENAGSILCSIHLAEQSHEVESKVLIGETFFQPSPWRLASILLGLLCGALCGVVMGMIIVFFKSKGKIQAELDWRRKHGQAELRDARKHAVEVTLDPETAHPKLCVSDLKTVTHRKAPQEVPHSEKRFTRKSVVASQGFQAGKHYWEVDVGQNVGWYVGVCRDDVDRGKNNVTLSPNNGYWVLRLTTEHLYFTFNPHFISLPPSTPPTRVGVFLDYEGGTISFFNTNDQSLIYTLLTCQFEGLLRPYIQHAMYDEEKGTPIFICPVSWG 83 Cynomolgus macaque BTNL8 aa MALMLSLVLSLLKLGSGQWQVFGPNKAVQALVGEDAAFSCFLSPKTNAEAMEVRFFRGQRFSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAGGRLSLRLENITVLDAGLYGCWIGSQS SYYQEATWELQVSALGSVPLISIVGYVDRGIQLLCRSSGWFPRPMAKWKGPQGQDLSADSRTNMNMHGLFDVELSLTVQENAGSVSCSMQHAHLSQEVESRVQIGDTFFQPVSWYLATKVLGILCCGLFFGIVGLKIFFSKFQGKIQAELDWRRKHRQAELRDARKHAVEVTLDPETAHPQLCVSDLKTVTHRKAHQDMPPSEKRFIRKSVVASQNFQAGRHYWEVDVGHNQRWYVGVCRDGVSRRKKRVTLSPNHGYWVLGMNAENLYFTLNPHFISLCPRTPLTQIGVFLDYECGTVSFFNINDQSLIYTLTCRFEGLLRPYIEYPSHNEQNGTPIVICPVTQESERKVSWQSASAIPEASNSEFSSQATTPFLPGSDA 84 Bicistronic construct nt

References : Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference.

Figure 1 : Identification of anti- BTNL8 mAbs . A. Screening cascade of anti-BTNL8 mAbs from mouse immunization to mAb sequencing. B. Bar graph showing the number of pure lines per affinity ( _KD ) range as measured on Luminex during primary hit selection. C. Stacked bar graph showing the number of anti-BTNL8 fusion tumor supernatant hits from primary screening that stained positive for the indicated cell line (MFI _fusion > MFI _HCM ) or negative (MFI _fusion < MFI _HCM ). Figure 2. Anti- BTNL8 fusion tumor supernatant based screening of Vγ4Vδ1 TCR reporter cells. A. Sketch illustrating Vγ4Vδ1 TCR reporter assay in which JRT3-Vγ4Vδ1-202 reporter cells were compared to HEK-BTNL8/BTNL3 cells (ratio) with or without 50 µL of anti-BTNL8 or irrelevant fusion tumor supernatant (control (Ctrl)). 1:1) co-culture for 16 h, resulting in BTNL8/BTNL3-mediated activation, as depicted by up-regulation of CD69 and NF-AT-GFP reporter and down-regulation of TCRVδ1 at the plasma membrane of reporter cells. B. Dot plots (top) and histograms (middle and bottom) of flow cytometry modulating JRT3-Vγ4Vδ1-202 reporter cell activation in the presence of HEK-BTNL8/BTNL3 and anti-BTNL8 or control fusion tumor supernatants Examples of metrology patterns. C. Stacked bar graph showing changes in the expression of NF-AT-GFP reporter, CD69 and TCRVδ1 at the plasma membrane of JRT3-Vγ4Vδ1-202 reporter cells induced in the presence of 47 anti-BTNL8 fusion tumor supernatants , these anti-BTNL8 fusion tumor supernatants were found to modulate reporter activation during primary hit screening. Figure 3. Anti- BTNL8 mAb enhances lysis of Vδ1 T cells . Vδ1 T cell lines were expanded from PBMCs of 3 healthy donors (see Materials and Methods) and grown in the presence of anti-CD107ab antibody and Golgistop with or without anti-BTNL8 or control fusion tumor supernatants Co-culture with HL-60 target cells using a 1:1 effector:target (E:T) ratio at 37°C. After 4 hours, cells were harvested, stained and analyzed on flow cytometry. A. BTNL8 expression in the HL-60 leukemia cell line as assessed on flow cytometry. B. Demonstrates degranulation of Vδ1 T cells in Vδ1 T cells cultured against HL60 myeloid leukemia cells alone or in the presence of a control isotype mAb (control) or anti-Vδ1 TCR mAb (positive control) or a representative reference anti-BTNL8 antibody ( % of CD107ab+ cells). Figure 4. Anti- BTNL8 mAb inhibits activation of Vγ4Vδ1 TCR reporter cells. In the presence of chimeric anti-BTNL8 mAb or corresponding isotype control, NF-AT-GFP reporter ( A ), CD69 ( B ) and TCRVδ1 ( C ) expression at the plasma membrane of JRT3-Vγ4Vδ1 TCR reporter cells co-cultured with HEK-BTNL3/BTNL8. The dose response of this inhibition as shown by _IC50 / _EC50 assessed for each mAb and each readout. Figure 5. Anti- BTNL8 mAb inhibits activation of primary Vγ2/3/4 cells. A. CD25 in Vγ2/3/4+Vδ1+ and Vγ2/3/4-Vδ1 assessed by FACS when Vδ1 T cells expanded from HD PBMC were co-cultured with HEK-pLV-null or HEK-BTNL3/BTNL8 Expression on +T cells. B. Effect of anti-BTNL8 mAb on Vγ2/3/4+Vδ1+ cell activation when co-cultured with HEK-BTNL3/BTNL8.

Claims (16)

An antibody specific for human BTNL8 (BTNL8), characterized in that the antibody has one or more of the following properties: i. It binds to human BTNL8/BTNL3 dimers as expressed in cell lines, such as HEK-293T cells, and/or ii. It does not bind to cell lines expressing BTNL3 but not BTNL8, such as HEK-293T cell line expressing BTNL3. An antibody specific for human BTNL8 (BTNL8) characterized in that the antibody binds to human _BTNL8 -Fc with a KD below 10 pM, eg, as measured by a Luminex assay. The anti-BTNL8 antibody of claim 1 or 2, wherein the anti-BTNL8 antibody has at least one of the following properties: i. It inhibits the activation of Vδ1 TCR-bearing T cells (Vδ1 T cells), ii. It inhibits the lytic function of activated Vδ1 T cells, and/or iii. It inhibits the production of cytokines by activated Vδ1 T cells. The anti-BTNL8 antibody of claim 1 or 2, wherein the anti-BTNL8 antibody has one or more of the following properties: i. It inhibits activation of Vγ4Vδ1 TCR-bearing T cells, typically as determined by a Vγ4Vδ1 TCR reporter cell assay; and/or ii. It inhibits the lytic function of activated Vδ1 T cells, generally it inhibits the degranulation of Vδ1 T cells against HL-60 cells, as determined in an in vitro degranulation cell assay. The anti-BTNL8 antibody of claim 1 or 2, wherein the anti-BTNL8 antibody inhibits activation of T cells bearing the Vγ4Vδ1 TCR with an _IC50 of less than 1.1 nM, as measured by CD69 expression at the plasma membrane as a readout. The anti-BTNL8 antibody of claim 1 or 2, which cross-reacts with cynomolgus monkey BTNL8. The anti-BTNL8 antibody of claim 1 or 2, which competes for binding to BTNL8 with at least one of the following reference murine antibodies: i. Reference murine antibody mAb X1 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 variable area; ii. Reference murine antibody mAb X2 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:4 variable area; iii. Reference murine antibody mAb X4 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8 variable area; or iv. Reference murine antibody mAb X5 comprising (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO:10 variable area. As in the anti-BTNL8 antibody of claim 1 or 2, its i. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X1 comprising SEQ ID NOs: 11 to 16, respectively; ii. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X2 comprising SEQ ID NOs: 17 to 22, respectively; iii. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X4 comprising SEQ ID NOs: 29 to 34, respectively; or iv. H-CDR1, H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of mAb X5 comprising SEQ ID NOs: 35 to 40, respectively. The anti-BTNL8 antibody of claim 1 or 2, which is an antibody comprising: i. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:1 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:2; ii. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:3 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:4; iii. A heavy chain wherein the VH region is at least 90% identical to SEQ ID NO:7 and a light chain wherein the VL region is at least 90% identical to SEQ ID NO:8; or iv. A heavy chain in which the VH region is at least 90% identical to SEQ ID NO:9 and a light chain in which the VL region is at least 90% identical to SEQ ID NO:10. The anti-BTNL8 antibody of claim 1 or 2, which is a human, chimeric or humanized antibody. A nucleic acid molecule encoding the heavy and/or light chain of the anti-BTNL8 antibody of any one of claims 1 to 10. A host cell comprising the nucleic acid of claim 11. A use of the anti-BTNL8 antibody of any one of claims 1 to 10 for the manufacture of a medicament. Use of an anti-BTNL8 antibody as claimed in any one of claims 1 to 10 in the manufacture of a medicament for the treatment of an inflammatory disorder, such as intestinal inflammation, in a subject. A use of the anti-BTNL8 antibody according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of an inflammatory disorder selected from the group consisting of inflammatory bowel disease (IBD), in particular Crohn's disease ), celiac disease or ulcerative colitis. A pharmaceutical composition comprising the anti-BTNL8 antibody of any one of claims 1 to 10 and at least one pharmaceutically acceptable carrier.

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