WO2021175954A1

WO2021175954A1 - Antibodies having specificity for btnl8 and uses thereof

Info

Publication number: WO2021175954A1
Application number: PCT/EP2021/055394
Authority: WO
Inventors: Carla CANO; Kieu Suong LE; Daniel Olive
Original assignee: Imcheck Therapeutics Sas; INSERM (Institut National de la Santé et de la Recherche Médicale); Centre National De La Recherche Scientifique - Cnrs -; Institut Jean Paoli & Irene Calmettes; Université D'aix-Marseille
Priority date: 2020-03-04
Filing date: 2021-03-03
Publication date: 2021-09-10
Also published as: TW202200617A

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 HAVING SPECIFICITY FOR BTNL8 AND USES THEREOF TECHNICAL FIELD

The present invention relates to anti-BTNL8 antibodies that bind to BTNL8 and inhibit V51 T cell cytotoxicity. Such antibodies are useful in particular for the treatment of inflammatory diseases.

BACKGROUND

White blood cells are cells of the immune system involved in defending the body against pathogens. In addition to conventional MHC class l-restricted CD8+ CTL and NK cells, other unconventional T cells, notably gd T cells, display the same sensitivity and cytolytic power as NK and T cells.

Butyrophilin and butyrophilin-like proteins (BTN/BTNL) are a family of immunoglobulin superfamily members that influence immunity, such as T cell selection, as well as 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 gut epithelial cells jointly induce selective TCR-dependent responses of human colonic Vy4/V51 cells.

Melandri et al (Nat Immunol. 2018, 19(12): 1352-1365) suggested a conserved mechanism whereby the regulation of gd intraepithelial lymphocytes (lELs) by Btnl or BTNL porteins is mediated by an “interacting chain” (Btnl6 or BTNL3) coupled to a supporting chain (Btnl1 or BTNL8) that jointly determine biological activity.

WO2019/234136 further reports a direct interaction of BTNL3/BTNL8 heterodimers with certain recombinant g4 containing TCRs, including g4d1 TCRs.

WO2019/053272 also reports compositions and methods useful in treating inflammation in the gut by modulating gd T cells, and for example Vy4+ cells.

WO2019/057933 discloses antibodies having specificity for BTN2 and their uses in methods for treating autoimmune and inflammatory disorders.

To date, there is still a need to identify treatment for inflammatory diseases, such as inflammatory diseases caused by inflammation in the gut, including without limitation, inflammatory bowel diseases, coeliac disease, Crohn’s disease or ulcerative colitis. More generally, there is a need to identify new suppressive agents and/or methods to inhibit immune response in a patient in need thereof.

The present disclosure discloses that certain anti-BTNL8 antibodies can inhibit V51 T cell activity, in particular V51 T cell cytotoxicity. Such antibodies are therefore useful in particular for the treatment of inflammatory diseases.

SUMMARY

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

In specific embodiments, such antibody having specificity for human BTNL8 (BTNL8), binds to human BTNL8-Fc with a K_D below 10 pM, for example as measured by Luminex assay.

In preferred embodiments, an anti-BTNL8 antibody of the present disclosure has at least one of the following properties: i. it inhibits the activation of T cells bearing a V51 TCR (V51 T cells), ii. it inhibits the cytolytic function of activated V51 T cells, and/or, iii. it inhibits the production of cytokines by activated V51 T cells.

In specific embodiments, an anti-BTNL8 antibody of the present disclosure has one or more of the following properties: i. it inhibits the activation of Vy4V61 TCR bearing T cells, typically as determined by Vy4V51 TCR reporter cell assay; and/or, ii. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay;

In specific embodiments, the anti-BTNL8 antibody of the present disclosure, further advantageously cross-reacts with cynomolgus BTNL8. Examples of said anti-E3TNL8 antibodies include the following reference murine antibodies: i. The 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; ii. The 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; iii. The 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; iv. The 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; or, v. The 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.

Also part of the disclosure are anti-E3TNL8 antibodies which competes for binding to BTNL8 with at least one of the above defined reference antibodies.

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

The anti-BTNL8 antibody of any one of the preceding claims, which is an antibody comprising either, i. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:1 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:2; ii. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:3 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:4; iii. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:5 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:6; iv. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:7 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:8; or, v. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:9 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:10.

Typically, said anti-BTNL8 antibody of the disclosure may be a human, chimeric or humanized antibody.

In another aspect, the disclosure relates to nucleic acid molecule which encodes a heavy chain and/or a light chain of the anti-BTNL8 antibody as defined above.

The disclosure further provides a host cell comprising any of the nucleic acid molecules encoding the anti-BTNL8 antibody as above-mentioned.

The disclosure also relates to the anti-BTNL8 antibody as described herein, for use a medicament, for example in treating inflammatory disorders, for example, inflammation in the gut of a subject. Preferred examples of such inflammatory disclosers include without limitation inflammatory bowel disease (IBD), in particular Crohn’s disease, celiac disease or ulcerative colitis.

The disclosure further relates to a pharmaceutical composition comprising said anti- BTNL8 antibody of the disclosure, and at least a pharmaceutically acceptable carrier. DETAILED DESCRIPTION Definitions

As used herein the term “BTNL8” has its general meaning in the art and refers to human BTNL8 polypeptide including BTNL8 of SEQ ID NO:81.

SEQ ID NO:81: Homo sapiens butyrophilin like 8 (BTNL8)

MALMLSLVLSLLKLGSGQWQVFGPDKPVQALVGEDAAFSCFLSPKTNAEAMEVRFFRG

QFSSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAEGRISLRLENITVLDAGLYGCRISS

QSYYQKAIWELQVSALGSVPLISITGYVDRDIQLLCQSSGWFPRPTAKWKGPQGQDLST

DSRTNRDMHGLFDVEISLTVQENAGSISCSMRHAHLSREVESRVQIGDTFFEPISWHLAT

KVLGILCCGLFFGIVGLKIFFSKFQWKIQAELDWRRKHGQAELRDARKHAVEVTLDPETA

HPKLCVSDLKTVTHRKAPQEVPHSEKRFTRKSVVASQSFQAGKHYWEVDGGHNKRWR

VGVCRDDVDRRKEYVTLSPDHGYWVLRLNGEHLYFTLNPRFISVFPRTPPTKIGVFLDYE

CGTISFFNINDQSLIYTLTCRFEGLLRPYIEYPSYNEQNGTPIVICPVTQESEKEASWQRAS

Al PETSNSESSSQATTPFLPRGEM

As used herein the term "antibody" or "immunoglobulin" have the same meaning and will be used equally in the present disclosure.

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

The term “antibody” as used herein 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 for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results. Additionally, for the embodiment in which the bispecific molecule is multi-specific, the molecule can further include a third binding specificity, in addition to the first and second target epitope.

In natural antibodies, two heavy chains are linked to each other by di-sulfide bonds and each heavy chain is linked to a light chain by a di-sulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The 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 both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains 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 (FcR).

The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.

The light and heavy chains of an immunoglobulin each have three CDRs, designated L- CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. According the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H- CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

In specific embodiments, an antibody provided herein is an antibody fragment, and more particularly any protein including an antigen-binding domain of an antibody 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 “specificity for BTNL8” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as a BTNL8. In other embodiments, it is intended to refer to an antibody that binds to the heterodimer BTNL8/BTNL3 as expressed in a cell line, for example HEK293T cell lines as described in the examples. In some embodiments, it is intended to refer to an antibody that (i) binds to human BTNL8/BTLN3 dimer as expressed in a cell line, for example HEK293T cell lines, and (ii) does not bind to a cell line expressing BTNL3 but not BTNL8, typically as described in the examples. In other embodiments, it binds to a BTNL8 recombinant polypeptide with a K_D 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 human BTNL8-Fc protein as assessed by Luminex based assessment of the affinity. More specifically, it binds to human BTNL8-Fc protein with a K_D below 100 pM, more preferably below 10 pM, as assessed by Luminex based assessment as described in the Examples.

In some embodiments, said antibody with specificity for BTNL8 further does not cross- react with BTNL3. An antibody that "cross-reacts with an antigen other than BTNL8" is intended to refer to an antibody that binds that antigen with a K_D 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" (for example BTNL3) is intended to refer to an antibody that binds to that antigen, with a K_D of 100 nM or greater, or a K_D of 1 mM or grater, or a K_D of 10 mM or greater. In certain embodiments, such antibodies do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays. Cross-reactivity may be tested either by affinity binding assay with the recombinant antigen, for example recombinant BTNL3-Fc, or with the cell expressing said antigen, for example, a cell line expressing BTNL3.

An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTNL8 is substantially free of antibodies that specifically bind to other antigens than BTNL8). An isolated antibody that specifically binds to BTNL8 may, however, have cross-reactivity to other antigens, such as related BTNL8 molecules from other species, for example cynomolgus BTNL8. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The phrases "an antibody recognizing an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen”.

The term "K_aSsoc" or "K_a", as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "Kdis" or "Kd," as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k_0ff to k_on (i.e. k_0ff/k_0n) and is expressed as a molar concentration (M). The K_D value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the K_D value (lower concentration) and thus the higher the affinity of the antibody. K_D values for antibodies can be determined using methods well established in the art. Preferred methods for determining the K_D values of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. A method for determining the K_D of an antibody is by using surface plasmon resonance, or by using a biosensor system such as a Biacore^® (see also for detailed information regarding affinity assessment Rich RL, Day YS, Morton TA, 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 Octet® systems The Octet® platform is based on bio-layer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces - a layer of immobilized protein and an internal reference layer. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (Dl) is a direct measure of the change in optical thickness of the biological layer, when this shift is measured over a period of time and its magnitude plotted as a function of time, a classic association/dissociation curve is obtained. This interaction is measured in real-time, allowing to monitor binding specificity, association rate and dissociation rate, and concentration (see Abdiche et al. 2008 but also the details in the results). Affinity measurements are typically performed at 25 °C. Another method may be the Luminex assay as described in the Examples.

Specificity can further be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is a BTNL8 polypeptide). The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope.

As used herein, the term “identity” refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, then the respective molecules are identical at that position. The percentage of identity between 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. Generally, a comparison is made when two sequences are aligned to give maximum identity. The identity may be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA or CLUSTALW.

As used herein, a functional variant of a reference antibody according to the present disclosure exhibits functional properties that are substantially equal or superior to the corresponding functional properties of the reference antibody (e.g. any one of mAb X1 - X5). By substantially equal, it is herein intended that said 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 the anti-BTNL8 antibodies of the disclosure

In one aspect, an antibody having specificity for BTNL8 as per the present disclosure can also be further characterized in that it has at least one of the following properties:

- it inhibits the activation of T cells bearing a V51TCR (V51 T cells),

- it inhibits the cytolytic function of activated V51 T cells, and/or

- it inhibits the production of cytokines by activated V51 T cells.

As used herein, by “inhibiting the activation of T cells bearing a V51TCR”, it is meant that a significant decrease of the expression of a reporter gene under the control of NF-AT promoter is observed in a Vy4V51TCR reporter cell assay, using a T cell line derivative devoid of TCRap that is transduced to express a colon-derived Vy4V51TCR and said reporter gene in the presence of cell lines expressing BTNL8 and BTNL3. “inhibiting the activation of T cells bearing a V51TCR” may also be determined by measuring the decrease of CD69 expression at the plasma membrane or by measuring the inhibition of the downregulation of TCRV61 expression at the plasma membrane. An example of such reporter cell assay is described in more details in the Examples below.

In some embodiments, the antibodies of the present disclosure inhibit the activation of T cells bearing a Vy4V51TCR (Vy4V61 T cells) to a level that is substantially different to at least one of the reference antibodies : mAb X1, mAb X2, mAb X3, mAb X4, and mAb X5 as described below.

As used herein, by “inhibiting the cytolytic function of activated V51 T cells”, it is meant a significant decrease of the cytolytic function of activated V51 T cells is observed when compared to control activated V51 T cells (with control isotype). In specific embodiments, the anti-BTNL8 antibodies of the present disclosure inhibits the cytolytic function of activated V51 T cells to a level that is substantially equal or superior to the basal V51 T cell degranulation against HL-60 cell line as described below. In more specific embodiments, the level of the cytolytic function of activated V51 T cells with the tested anti-BTNL8 antibody is reduced to at least 5 %, more specifically at least 10% and preferably at least 15%, as compared to a control isotype antibody.

Hence, the anti-BTNL8 antibodies of the present disclosure having such advantageous properties as above defined can be screened among anti-BTNL8 antibodies using the cellular assays as described in the Examples and in particular, assays for determining the modulation of the degranulation of V51 T cells against BTNL8-expressing cancer cell lines such as the HL-60 myelogenous leukaemia cell line.

In some embodiments, the antibodies of the present disclosure inhibits the cytolytic function of activated V51 T cells to a level that is substantially equal or superior to at least one of the reference antibodies : mAb X1, mAb X2, mAb X3, mAb X4, and mAb X5 as described below.

As used herein, by “inhibiting the production of cytokines by activated V51 T cells”, it is meant that a significant decrease of the production of cytokines, typically TNFa and/or IFNy is observed compared to the production of cytokines from activated V51 T cells in the presence of a control isotype antibody.

Hence, in preferred embodiments, said anti-BTNL8 antibody of the present disclosure exhibits at least one or more of the following functional properties: i. it has specificity for BTNL8, in particular binding to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with an EC50 below 50 pg/mL and more specifically below 40 pg/mL or with a KD, as measured by surface plasmon resonance (SPR) (typically at 25 °C), or Luminex assay (typically as illustrated in the Examples) or Octet® (Abdiche et al. 2008) of 10 nM or less, and/or it has an about 10:1, about 20: 1 , about 50:1, about 100:1, 10.000:1 or greater ratio of affinity in binding BTNL8 versus non-specific binding; and/or ii. it binds to human BTNL8/BTNL3 dimer as expressed in a cell line, for example HEK293T cells, preferably stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell line expressing BTNL3, for example as illustrated in the Examples; iv. it modulates the activation of Vy4V61 TCR bearing T cells, typically as determined by a Vy4V51 TCR reporter cell assay, for example as illustrated in the Examples, typically either:

• it inhibits the activation of Vy4V61 TCR bearing T cells with an IC50 below 1 nM, typically between 0.1 and 1 nM, or below 0.2 pg/ml, typically between 0.01 and 0.2 pg/ml as measured with NF-AT-GFP gene reporter as a readout,

• it inhibits the activation of Vy4V61 TCR bearing T cells with an IC50 below 1.1 nM, typically between 0.3 and 1.1 nM or between 0.05 and 0.17 pg/ml as measured with CD69 expression at the plasma membrane as a readout, and/or

• it inhibits the TCR downregulation of Vy4V61 TCR bearing T cells with an EC50 below 1.4, typically between 0.5 and 1.4 nM or between 0.05 and 0.21 pg/ml as measured with TCRV61 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay, for example as illustrated in the Examples; vi. it inhibits cytokines (e.g.: TNFa) production from activated V61 T cells, typically assessed as illustrated in the Examples, and/or, vii. it cross-reacts with cynomolgus BTNL8, viii. it binds to human BTNL8, in particular to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with a K_D below 10 nM, particularly below 1 nM, as measured by BLI (typically as illustrated in examples).

In various embodiments, the anti-BTNL8 antibody of the disclosure may 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 “silent” antibody refers to an antibody that exhibits no or low ADCC activity as measured in an in vitro ADCC activity assay measuring cell lysis of target cells.

In one embodiment, the term “no or low ADCC activity” means that the silent antibody exhibit an ADCC activity that is at below 50%, for example below 10% of the ADCC activity that is observed with the corresponding wild type (non-silent) antibody for example with a wild type human lgG1 antibody. Preferably, no detectable ADCC activity is observed in an in vitro ADCC activity assay with a silent antibody as compared to a control Fab antibody.

Silenced effector functions can be obtained by mutation in the Fc constant part of the antibodies 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 silent lgG1 antibodies comprise mutations reducing ADCC at positions 234, 235 and/or 331 in the lgG1 Fc amino acid sequence (EU numbering). Another silent lgG1 antibody comprises the N297A mutation, which results in aglycosylated or non-glycosylated antibodies.

In some embodiments, the anti-BTNL8 antibody of the present disclosure is selected from the group consisting of Fab, F(ab')2, Fab' and scFv. As used herein, the term “Fab” denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a di-sulfide bond. The term “F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a di-sulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin. The term “Fab¹ “ refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a di-sulfide bond of the hinge region of the F(ab')2. A single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the disclosure includes CDRs that are held in appropriate conformation, preferably by using gene recombination techniques. Reference antibodies mAbs X1-X5

Antibodies of the disclosure include the reference murine monoclonal anti-BTNL8 antibodies isolated and structurally characterized by their variable heavy and light chain amino acid sequences as described in the Table 1 below. In one preferred embodiment, the reference murine monoclonal antibodies are mAb X1, mAb X2, mAb X4, mAb X5 and not mAb X3.

Table 1 : Variable heavy and light chain amino acid sequences of murine reference antibodies of the disclosure

Examples of the amino acid sequences of the VH CDR1s (also called HCDR1), VH CDR2s (also called HCDR2), VH CDR3s (also called HCDR3), VL CDR1s (also called LCDR1), VL CDR2s (also called LCDR2), VL CDR3s (also called LCDR3) of some antibodies according to the disclosure are shown in Table 2.

In Table 2, the CDR regions of some antibodies of the present disclosure are delineated using the Kabat system. For the ease of reading, the CDR regions are called hereafter HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 respectively. Table 2: CDR regions of reference murine antibodies according to Kabat definition

In specific embodiments, the isolated anti-BTNL8 antibody according to the disclosure comprises either: (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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:14, LCDR2 of SEQ ID NO:15 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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:20, LCDR2 of SEQ ID NO:21 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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:26, LCDR2 of SEQ ID NO:27 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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:32, LCDR2 of SEQ ID NO:33 and 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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:38, LCDR2 of SEQ ID NO:39 and LCDR3 of SEQ ID NO:40; wherein said anti-BTNL8 antibody has specificity for BTNL8.

In a preferred embodiment, the isolated anti-BTNL8 antibody according to the disclosure has specificity for BTNL8 and comprises either:

(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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:14, LCDR2 of SEQ ID NO:15 and LCDR3 of SEQ ID NO:16;

(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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:32, LCDR2 of SEQ ID NO:33 and 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 a variable light chain polypeptide comprising LCDR1 of SEQ ID NO:38, LCDR2 of SEQ ID NO:39 and LCDR3 of SEQ ID NO:40;

In other specific embodiments, the isolated anti-BTNL8 antibody according to the disclosure comprises either:

(a) a variable heavy chain polypeptide comprising VH of SEQ ID NO:1 and a variable light chain polypeptide VL of SEQ ID NO:2;

(b) a variable heavy chain polypeptide comprising VH of SEQ ID NO:3 and a variable light chain polypeptide VL of SEQ ID NO:4; (c) a variable heavy chain polypeptide comprising VH of SEQ ID NO:5 and a variable light chain polypeptide VL of SEQ ID NO:6;

(d) a variable heavy chain polypeptide comprising VH of SEQ ID NO:7 and a variable light chain polypeptide VL of SEQ ID NO:8; or

(e) a variable heavy chain polypeptide comprising VH of SEQ ID NO:9 and a variable light chain polypeptide VL of SEQ ID NO:10; wherein said anti-BTNL8 antibody has specificity for BTNL8.

In another preferred embodiments, the isolated anti-BTNL8 antibody according to the disclosure has specificity for BTNL8 and comprises either:

(b) a variable heavy chain polypeptide comprising VH of SEQ ID NO:3 and a variable light chain polypeptide VL of SEQ ID NO:4;

(c) a variable heavy chain polypeptide comprising VH of SEQ ID NO:7 and a variable light chain polypeptide VL of SEQ ID NO:8; or

(d) a variable heavy chain polypeptide comprising VH of SEQ ID NO:9 and a variable light chain polypeptide VL of SEQ ID NO:10; wherein said anti-BTNL8 antibody has specificity for BTNL8.

Functional variant antibodies

In yet another embodiment, a functional variant antibody of the disclosure has full length heavy and light chain amino acid sequences; or variable region heavy and light chain amino acid sequences, or all 6 CDR regions amino acid sequences that are homologous or more specifically identical to the corresponding amino acid sequences of the reference antibody mAb X1, X2, X3, X4 or X5 as described above, in particular in Tables 1 and 2, and wherein such functional variant antibodies exhibit the desired functional properties of any of said reference antibody mAb X1 - mAb X5.

A functional variant of the reference mAb X1-X5 antibody, notably a functional variant of a VL, VH, or CDR used in the context of a monoclonal antibody of the present disclosure still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the affinity (typically assessed by KD as measured by Luminex assay, and/or the specificity/selectivity of the parent antibody ( e.g.\ any one mAb X1 to mAb X5 antibody) and in some cases such a monoclonal antibody of the present disclosure may be associated with greater affinity, selectivity and/or specificity than the parent Ab (e.g. any one of mAb X1 to mAb X5 antibody).

Desired functional properties of the reference mAb X1 - mAb X5 may be selected from the group consisting of: i. it has specificity for BTNL8, in particular binding to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with an EC50 below 50 pg/mL and more specifically below 40 pg/mL or with a KD, as measured by surface plasmon resonance (SPR) (typically at 25 °C), or Luminex assay (typically as illustrated in the Examples) or Octet® (Abdiche et al. 2008) of 10 nM or less, and/or it has an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity in binding BTNL8 versus non-specific binding; and/or ii. it binds to human BTNL8/BTNL3 as expressed in a cell line, for example HEK293T cells, preferably stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell line expressing BTNL3, for example as illustrated in the Examples; iv. it modulates the activation of Vy4V61TCR bearing T cells, typically as determined by a Vy4V51TCR reporter cell assay, for example as illustrated in the Examples, typically, either:

• it inhibits the activation of Vy4V61 TCR bearing T cells with an IC50 below 1 nM, typically between 0.1 and 1 nM, or below 0.2 pg/ml, typically between 0.01 and 0.2 pg/ml, as measured with NF-AT-GFP gene reporter as a readout,

• it inhibits the activation of Vy4V61 TCR bearing T cells with an IC50 below 1.1 nM, typically between 0.3 and 1.1 nM, or below 0.17 pg/ml, typically between 0.05 and 0.17 pg/ml, as measured with CD69 expression at the plasma membrane as a readout, and/or

• it inhibits the TCR downregulation of Vy4V61 TCR bearing T cells with an EC50 below 1.4, typically between 0.5 and 1.4 nM, or below 0.21 pg/ml, typically between 0.05 and 0.21 pg/ml, as measured with TCRV61 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay, for example as illustrated in the Examples; vi. it inhibits cytokines (e.g.: TNFa) production from activated V61 T cells, typically assessed as illustrated in the Examples, and/or, vii. it cross-reacts with cynomolgus BTNL8. viii. it binds to human BTNL8, in particular to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with a K_D below 10 nM, particularly below 1 nM, as measured by BLI (typically as illustrated in examples).

For example, the disclosure relates to functional variant antibodies of mAb X1-mAb X5, preferably functional variant antibodies of mAb X1, X2, X4, and X5, comprising a variable heavy chain (VH) and a variable light chain (VL) sequences where the CDR sequences, i.e. the 6 CDR regions; HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 share at least 60, 70, 90, 95 or 100 percent sequence identity to the corresponding CDR sequences of at least one antibody of mAb X1-mAb X5, as shown in Table 2, wherein said functional variant antibody specifically binds to BTNL8, and the antibody exhibits at least one of the following functional properties: i. it has specificity for BTNL8, in particular binding to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with an EC50 below 50 pg/mL and more specifically below 40 pg/mL or with a KD, as measured by surface plasmon resonance (SPR) (typically at 25 °C), or Luminex assay (typically as illustrated in the Examples) or Octet® (Abdiche et al. 2008) of 10 nM or less, and/or it has an about 10:1, about 20: 1 , about 50:1, about 100:1, 10.000:1 or greater ratio of affinity in binding BTNL8 versus non-specific binding; ii. it binds to human BTNL8/BTNL3 dimer as expressed in a cell line, for example HEK293T cells, preferably stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell line expressing BTNL3, for example as illustrated in the Examples; iv. it modulates the activation of Vy4V61 TCR bearing T cells, typically as determined by a Vy4V51 TCR reporter cell assay, for example as illustrated in the Examples, typically, either:

• it inhibits the TCR downregulation of Vy4V61 TCR bearing T cells with an EC50 below 1.4, typically between 0.5 and 1.4 nM, or below 0.21 pg/ml, typically between 0.05 and 0.21 pg/ml, as measured with TCRV61 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay, for example as illustrated in the Examples; vi. it inhibits cytokines (e.g.: TNFa) production from activated V61 T cells, typically assessed as illustrated in the Examples, vii. it cross-reacts with cynomolgus BTNL8, and/or, viii. it binds to human BTNL8, in particular to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with a KD below 10 nM, particularly below 1 nM, as measured by BLI (typically as illustrated in examples).

It further relates to functional variant antibodies of mAb X1-mAb X5, preferably functional variant antibodies of mAb X1, X2, X4, and X5, comprising a heavy chain variable region and a light chain variable region that are at least 80%, 90%, or at least 95% or 100% identical to the corresponding heavy and light chain variable regions of any one of mAb X1-mAb X5 antibodies, as shown in particular in Table 1; the functional variant antibody specifically binds to BTNL8, and exhibits at least one of the following functional properties: i. it has specificity for BTNL8, in particular binding to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with an EC50 below 50 pg/mL and more specifically below 40 pg/mL or with a KD, as measured by surface plasmon resonance (SPR) (typically at 25 °C), or Luminex assay (typically as illustrated in the Examples) or Octet® (Abdiche et al. 2008) of 10 nM or less and/or it has an about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity in binding BTNL8 versus non-specific binding; ii. it binds to human BTNL8/BTNL3 as expressed in a cell line, for example HEK293T cells, preferably stably transduced with a lentiviral vector encoding human BTNL8 and human BTNL3, iii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell line expressing BTNL3, for example as illustrated in the Examples; iv. it modulates the activation of Vy4V61 TCR bearing T cells, typically as determined by a Vy4V51TCR reporter cell assay, for example as illustrated in the Examples, typically, either:

• it inhibits the TCR downregulation of Vy4V61 TCR bearing T cells with an EC50 below 1.4, typically between 0.5 and 1.4 nM, or below 0.21 pg/ml, typically between 0.05 and 0.21 pg/ml, as measured with TCRV61 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay, for example as illustrated in the Examples; vi. it inhibits cytokines (e.g.: TNFa) production from activated V61 T cells, typically assessed as illustrated in the Examples, vii. it cross-reacts with cynomolgus BTNL8, and/or, viii. it binds to human BTNL8, in particular to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with a K_D below 10 nM, particularly below 1 nM, as measured by BLI (typically as illustrated in examples).

The sequences of CDR variants may differ from the sequence of the CDRs of the parent/reference antibody sequences (as shown for example in Table 2) through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:

Aliphatic residues I, L, V, and M

Cycloalkenyl-associated 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

Small residues A, C, D, G, N, P, S, T, and V

Very small residues A, G, and S

Residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T

Flexible residues Q, T, K, S, G, P, D, E, and R

More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of the any one of mAbs X1- X5. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 1 1 and Extended Gap= I).

Suitable variants typically exhibit at least about 90%, for example 95% of identity to the parent polypeptide VH and VL sequences. According to the present disclosure a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence. According to the present disclosure a first amino acid sequence having at least 50% of identity with a second amino acid sequence means that the first sequence has 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% of identity with the second amino acid sequence.

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 which comprises a VH domain and a VL domain of an antibody derived from a non-human animal, a CH domain and a CL domain 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, said mouse/human chimeric antibody may comprise the VH and the VL domains of any one of mAb X1-mAb X5 reference antibodies.

In some embodiments, the functional variant is a humanized antibody. In specific embodiments, the antibody of the present disclosure is a humanized antibody which comprises the 6 CDRs of any one of the mAb X1-mAbX5 reference antibodies, for example as shown in Table 2. As used herein the term "humanized antibody" refers to antibodies in which the framework regions (FRs) have been modified to avoid potentially immune residues in human. For example, the humanized antibody comprises the FRs from a donor immunoglobulin of human species as compared to that of the parent immunoglobulin (for example murine CDRs).

Functional variant antibodies with mutant amino acid sequences can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the coding nucleic acid molecules, followed by testing of the encoded altered antibody for retained function (i.e., the functions set forth above) using the functional assays described herein.

Antibodies that cross-compete at least one of mAb X1-mAb X5 and/or that bind to the same epitope as one of mAbX1-mAbX5

Additional antibodies with similar advantageous properties of at least one of the reference antibodies mAb X1-mAb X5 as disclosed herein can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of), in a statistically significant manner with any one of the reference antibodies mAb X1-mAb X5 as described above, in standard BTNL8 binding assays.

Test antibody may first be screened for their binding affinity to BTNL8, for example from human recombinant antibody libraries using for example phage display technologies or from transgenic mouse expressing human variable region antibodies immunized with BTNL8 antigens.

The ability of a test antibody to cross-compete with or inhibit the binding of antibodies of the present disclosure to human BTNL8 demonstrates that the test antibody can compete with that antibody for binding to human BTNL8; such an antibody may, according to non limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on human BTNL8 as the antibody with which it competes.

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

The selected antibodies can be further tested for the advantageous properties of mAb X1- mAb X5 in particular with respect to inhibition properties against activated Vy4V51 T cells.

Accordingly, in one embodiment, the disclosure provides an isolated antibody which compete for binding to at least one reference antibody of mAb X1-mAb X5, from binding to BTNL8, wherein said antibody exhibits at least one of the following properties: i. it has specificity for BTNL8, in particular binding to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with an EC50 below 50 pg/mL and more specifically below 40 pg/mL or with a KD, as measured by surface plasmon resonance (SPR) (typically at 25 °C), or Luminex assay (typically as illustrated in the Examples) or Octet® (Abdiche et al. 2008) of 10 nM or less, and/or it has an about 10:1, about 20: 1 , about 50:1, about 100:1, 10.000:1 or greater ratio of affinity in binding BTNL8 versus non-specific binding; and/or ii. it binds to human BTNL8/BTNL3 dimer as expressed in a cell line, for example HEK293T cells, preferably stably transduced with lentiviral vectors encoding human BTNL8 and human BTNL3, iii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell line expressing BTNL3, for example as illustrated in the Examples; iv. it modulates the activation of Vy4V61 TCR bearing T cells, typically as determined by Vy4V61 TCR reporter cell assay, for example as illustrated in the Examples, typically, either:

• it inhibits the TCR downregulation of Vy4V61 TCR bearing T cells with an EC50 below 1.4, typically between 0.5 and 1.4 nM, or below 0.21 pg/ml, typically between 0.05 and 0.21 pg/ml, as measured with TCRV61 expression at the plasma membrane as a readout, as illustrated in the Examples; v. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V61 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay, for example as illustrated in the Examples; vi. it inhibits cytokines (e.g.: TNFa) production from activated V61 T cells, typically assessed as illustrated in the Examples, vii. it binds to human BTNL8, in particular to human BTNL8 as expressed in a cell line, for example HEK293T cells stably transduced with a lentiviral vector encoding human BTNL8, as described in the examples, more specifically with a KD below 10 nM, particularly below 1 nM, as measured by BLI (typically as illustrated in examples), and/or, viii. it cross-reacts with cynomolgus BTNL8,

Typically, functional properties according to points (iv) to (viii) above of an antibody that compete for binding to BTNL8 with at least one of reference mAb X1 - X5 are substantially equal or superior to the corresponding functional properties of said corresponding reference antibody mAb X1 - X5, as described above. By substantially equal it is herein intended that the functional variant retains at least about 50%, 60%, 70%, 80%, 90%, 95% or 100% of the corresponding functional property of said reference mAb X1 to X5.

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

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

In a specific embodiment, the disclosure 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 that do not bind or cross-compete to the same epitope anti-BTNL8 antibodies mAb X2, X4 and X5.

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

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

Generation of transfectomas producing monoclonal antibodies

The antibodies of the present disclosure are produced by any techniques known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions.

Alternatively, antibodies of the present disclosure can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.

Accordingly, a further object of the disclosure relates to a nucleic acid molecule encoding an antibody according to the disclosure, typically the reference antibodies mAb X1-X5 or their functional variants. More particularly the nucleic acid molecule encodes a heavy chain or a light chain of an antibody of the present disclosure. More particularly the nucleic acid molecule comprises a VH or VL coding region having at least 70%, 80%, 90%, 95% or 100% of identity to the corresponding nucleic acid encoding heavy chain variable region (VH region) or light chain variable region (VL) of any one of the reference antibodies mAb X1-mAb X5, for example as disclosed in any one of SEQ ID NO 47-48 (mAb X1), SEQ ID NO:55-56 (mAb X2), SEQ ID NO:63-64 (mAb X3), SEQ ID NO:71-72 (mAb X4), SEQ ID NO:79-80 (mAb X5).

Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. So, a further object of the disclosure relates to a vector comprising a nucleic acid of the disclosure for producing the antibody. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukaemia virus, promoter and enhancer of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1 beta d2-4 and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance 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.

A further object of the present disclosure relates to a host cell which has been transfected, infected or transformed by a nucleic acid and/or a vector as described above. As used herein, the term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically the antibody encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

The nucleic acids of the disclosure may be used to produce an antibody of the present disclosure in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafter referred to as "YB2/0 cell"), and the like.

The present disclosure also relates to a method of producing a recombinant host cell expressing an antibody according to the disclosure, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present disclosure. The recombinant nucleic acid may be stably integrated in the genome of the host cell.

Antibodies of the present disclosure are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In some embodiments, a human chimeric antibody of the present disclosure can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as lgG1, lgG2, lgG3 and lgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

A humanized antibody of the present disclosure may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector having genes encoding (i) a heavy chain constant region and heavy chain variable framework regions identical to that of a human antibody and (ii) a light chain constant region and light chain variable framework regions identical to that of a human antibody, and expressing the genes by introducing the expression vector into suitable cell line. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist 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, e. g., 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 W09 1/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

The Fab of the present disclosure can be obtained by treating an antibody which specifically reacts with AMH with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a prokaryote or eucaryote (as appropriate) to express the Fab.

The F(ab')2 of the present disclosure can be obtained treating an antibody which specifically reacts with AMH with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a di-sulfide bond.

The Fab' of the present disclosure can be obtained treating F(ab')2 which specifically reacts with AMH with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

The scFv of the present disclosure can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv.

To generate a humanized scFv fragment, the well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

Engineered antibodies of the present disclosure further include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease 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 the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations 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 disclosure. 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 to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

Isotype and Fc engineering

The constant region of an antibody of the disclosure may be of any isotype. As used herein, the term “constant region” or “Fc region” is used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl- terminus of the IgG antibody wherein the numbering is according to the EU numbering system. The C-terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody or its corresponding codon deleted in the recombinant constructs. Accordingly, a composition of antibodies of the disclosure may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

The choice of isotype typically will be guided by the desired effector functions, such as ADCC silencing. Exemplary isotypes are IgGI, lgG2, lgG3, and lgG4. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of an antibody of the present disclosure may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from lgG1 to lgG2. Thus, the effector function of the antibodies of the present disclosure may be changed by isotype switching to, e.g., an IgGI, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In some embodiments, the antibody of the disclosure is a full-length antibody. In some embodiments, the full-length antibody is an IgGI antibody.

The antibodies of the disclosure may 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 functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. 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 abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by 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 approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

In other embodiments, the Fc region is modified to decrease the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody for an Fey receptor by modifying one or more amino acids. Such antibodies with decreased effector functions, and in particular decreased ADCC include silent antibodies.

In certain embodiments, the Fc domain of the lgG1 isotype is used. In some specific embodiments, a mutant variant of the lgG1 Fc fragment is used, e.g. a silent lgG1 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor.

In certain embodiments, the Fc domain of the lgG4 isotype is used. In some specific embodiments, a mutant variant of the lgG4 Fc fragment is used, e.g. a silent lgG4 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor.

Silenced effector functions can be obtained by mutation in the Fc constant part of the antibodies and have been described in the Art (Baudino et al., 2008; Strohl, 2009). Examples of silent lgG1 antibodies comprise the triple mutant variant lgG1 L247F L248E P350S. Examples of silent lgG4 antibodies comprise the double mutant variant lgG4 S241P L248E.

In certain embodiments, the Fc domain is a silent Fc mutant preventing 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 replacement of N314 by a glycine or an alanine.

In some embodiments, the full-length anti-BTNL8 antibody of the disclosure is an lgG4 antibody. In some embodiments, the anti-BTNL8 antibody is a stabilized lgG4 antibody. Examples of suitable stabilized lgG4 antibodies are antibodies wherein arginine at position 409 in a heavy chain constant region of human lgG4, which is indicated in the EU index as in Kabat et al. supra, is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in W02006033386) and/or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stabilized lgG4 antibodies are disclosed in WO2008145142.

In some embodiments, the antibody of the present disclosure does not comprise a Fc portion that induces antibody dependent cellular cytotoxicity (ADCC). The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human gamma heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, d, e and m for human antibodies), or a naturally occurring allotype thereof. Unless otherwise specified, the commonly accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (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 antibody of the present disclosure does not comprise an Fc domain capable of substantially binding to a FcyRIIIA (CD16) polypeptide. In some embodiments, the antibody of the present disclosure lacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises an Fc domain of lgG2 or lgG4 isotype. In some embodiments, the antibody of the present disclosure consists of or comprises a Fab, Fab', Fab'-SH, F (ab¹) 2, Fv, a diabody, single-chain antibody fragment, or a multi-specific antibody comprising multiple different antibody fragments.

In some embodiments, the antibody of the present disclosure is not linked to a toxic moiety. 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 abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551.

Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

A modification of the antibodies herein that is contemplated by the disclosure is pegylation or hesylation or related technologies. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1- C10) alkoxy- or aryloxy-poly ethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See for example, EP 0154 316 by Nishimura et al. and EP 0401 384 by Ishikawa et al.

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

In some embodiments, the disclosure also provides a multispecific antibody. Exemplary formats for the multispecific antibody molecules of the disclosure include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to BTNL8 and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-lg), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-lg™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically-linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so- called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., 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 (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies. In some embodiments, the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in W02008119353 and WO 2011131746 (both by Genmab A/S). In one exemplary method, described in WO 2008119353, a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising lgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present disclosure are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is the antibody of the present disclosure : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is the antibody of the present disclosure and the second antibody has a different binding specificity, or vice versa. The reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. Step d) may further comprise restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety. The following are exemplary embodiments of combinations of such asymmetrical mutations, optionally wherein one or both Fc-regions are of the lgG1 isotype.

Pharmaceutical compositions

In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing at least one antibody as disclosed herein, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies as described above. Pharmaceutical compositions disclosed herein also can be administered in combination therapy, i.e., combined with other agents.

For example, an antibody of the present disclosure may typically be combined with at least one anti-viral, anti-inflammatory or anti-microbial agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below in the section on uses of the antibodies of the disclosure.

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

Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

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

Preferably, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability 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.

Sterile injectable solutions may thus be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

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

Methods of use of the antibodies of the disclosure

The antibodies of the present disclosure have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.

The anti-BTNL8 antibodies of the disclosure are useful in particular in methods for treating, preventing or diagnosing E3TNL8/BTNL3-related disorders, or disorders related to altered expression of BTNL8/BTNL3, or disorders related to undesired activity of V51 T cells, in particular Vy4V51 T cells, in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of said anti-BTNL8 antibody of the present disclosure.

As used herein, a disorder related to undesired activity of V51 T cells, in particular Vy4V51 T cells, refer to any condition which is induced, enhanced or worsened in the presence of activated V51 T cells, in particular Vy4V51 T cells, and/or can be treated by reducing the functions of activated V51 T cells. This includes in particular conditions associated with or characterized by aberrant expression of BTNL8 or BTNL8/BTNL3 and/or diseases or conditions that can be treated by modulating BTNL8 and/or BTNL8-induced signaling activity in human blood cells e.g. by inhibiting the production of cytokines by activated V61 T cells and/or the cytolytic function of activated V51 T cells.

Thereby, the anti-BTNL8 antibodies of the disclosure may be used in methods for inhibiting the activation of V51 T cells, in particular in the presence of BTNL8/BTNL3 expressing cells, especially, in terms of cytokine secretion or cytolytic function, said methods comprising administering to the subject an inhibitory effective amount of said anti-BTNL8 antibody of the present disclosure.

An object of the present disclosure thus relates to a method of inhibiting an immune response in a subject, in particular inhibiting the cytolytic property of Vy4V61 T cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-BTNL8 antibody of the present disclosure.

In specific embodiments, the anti-BTNL8 antibodies of the disclosure are useful in particular for treating, preventing or diagnosing inflammatory disorders, for example inflammation in the gut of a subject.

The disclosure also pertains to the methods of manufacturing a medicament for use in the treatment of inflammatory conditions, said medicament comprising an anti-BTNL8 antibody of the present disclosure as described in the previous sections.

Examples of inflammatory diseases which may be treated include but are not limited to inflammatory bowel diseases, irritable bowel syndrome, diverticulitis, celiac disease, Crohn’s disease, ulcerative colitis, thyroiditis, metabolic disorders, immune related disorders, autoimmune disorders, transplantation rejection, post-traumatic immune response, graft-versus-host disease, ischemia, stroke, and infectious diseases.

In specific embodiments, the antibodies of the present disclosure are used for treating a disorder of the gastro-intestinal system, and/or associated to inflammation of the gut. Said disorder of the gastro-intestinal system include without limitation, inflammatory disorders of the gastrointestinal system and microbial infection of tissues of the gastrointestinal system.

The antibodies of the disclosure may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant to or in combination to, other drugs e.g. immunosuppressive or immunomodulating agents or other anti-inflammatory agents, e.g. for the treatment or prevention of diseases mentioned above. Typically, said anti-inflammatory agent may include without limitation, an anti-inflammatory cytokine, said cytokine being optionally selected from interleukin 10 (IL-10), interleukin 22 (IL-22). In other embodiment, said anti-inflammatory agent may include without limitation a steroid, for example a glucocorticoid, prednisone, hydrocortisone or an immunomodulator, including an anti-TNFa antibody or an anti-IL-17 antibody.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the antibody of the present disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody of the present disclosure 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. The efficient dosages and dosage regimens for the antibody of the present disclosure depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the antibody of the present disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present disclosure will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to treat inflammatory disorders, for example, be evaluated in an animal model system predictive of efficacy in treating inflammatory disorders. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to inhibit induction of immune response by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease immune or inflammatory response, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present disclosure is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance 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 for a therapeutically effective amount of an antibody of the present disclosure 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, for example about 0.5-2 mg/kg.

In accordance with the foregoing the present disclosure provides in a yet further aspect:

A method as defined above comprising co-administration, e.g., concomitantly or in sequence, of a therapeutically effective amount of an anti-BTNL8 antibody of the disclosure, and at least one second drug substance, said second drug substance being an anti-viral, anti-inflammatory, or anti-microbial agent, e.g. as indicated above.

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

Accordingly, in one aspect, the disclosure further provides methods for detecting the presence of BTNL8, or BTNL8 expressing cells (e.g., human BTNL8 antigen) in a sample, or measuring the amount of BTNL8, comprising incubating the sample, and a control sample, with an antibody of the disclosure, which specifically binds to BTNL8, under conditions that allow for formation of a complex between the antibody and BTNL8. The formation of a complex is then detected, wherein a difference in complex formation between the sample compared to the control sample is indicative of the presence of BTNL8 in the sample.

Also, within the scope of the present disclosure are kits consisting of the compositions (e.g., humanized antibodies of any one of the reference antibodies mAb X1 - X5) disclosed herein and instructions for use. The kit can further contain a least one additional reagent, or one or more additional antibodies or proteins. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. The kit may further comprise tools for diagnosing whether a patient belongs to a group that will respond to an anti-BTNL8 antibody treatment, as defined above.

The disclosure will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

LEDENDS OF THE FIGURES

Figure 1 : Identification of anti-BTNL8 mAb. A. Screening cascade of anti-BTNL8 mAbs from mice immunization to mAb sequencing. B. Bar chart shows the number of clones per affinity (KD) range as measured on Luminex during primary hit selection. C. Stacked bar chart show the number of anti-BTNL8 hybridoma supernatant hits from primary screening that were positive (MFIhybridoma > MFIHCM) or negative (MFIhybridoma < MFIHCM) for staining of the indicated cell lines.

Figure 2. Vy4V51TCR reporter cell-based screening of anti-BTNL8 hybridoma supernatants. A. Cartoon illustrates Vy4V51TCR reporter assay in which JRT3-Vy4V51- 202 reporter cells are co-cultured with HEK-BTNL8/BTNL3 cells (ratio 1:1) with or without 50 pl_ anti-BTNL8 or irrelevant hybridoma supernatant (Ctrl) for 16h, leading to BTNL8/BTNL3-mediated activation, as depicted by CD69 and NF-AT-GFP reporter upregulation and TCRV51 downregulation at the plasma membrane of reporter cells. B. Dot plots (top) and histograms (middle and bottom) are examples of flow cytometry patterns of modulation of JRT3-Vy4V51-202 reporter cell activation in presence of HEK- BTNL8/BTNL3 and anti-BTNL8 or Ctrl hybridoma supernatants. C. Stacked bar chart show the variation of the expressions of the NF-AT-GFP reporter, CD69 and TCRV51 at the plasma membrane of JRT3-Vy4V51-202 reporter cells induced in the presence of 47 anti-BTNL8 hybridoma supernatants found to modulate reporter activation during primary hit screening. Figure 3. Anti-BTNL8 mAb enhance the cytolytic function of V81 T cells. V51 T cells were expanded from PBMCs of 3 healthy donors (see Material and Methods), and co cultured at 37°C with HL-60 target cells using and effector: target (E:T) ratio of 1:1, in presence of anti-CD107ab antibodies and Golgistop, with or without anti-E3TNL8 or Ctrl hybridoma supernatants. After 4 hours, cells were collected, stained and analyzed on flow cytometry. A. BTNL8 expression in HL-60 leukemia cell line as assessed on flow cytometry. B. Contour plots showing V51 T cell degranulation (%CD107ab+ cells) among V51 T cells cultured alone, or against HL-60 myelogenous leukemia cells in presence of a control isotype mAb (Ctrl), or anti-V51TCR mAb (positive control) or a representative reference anti-BTNL8 antibody.

Figure 4. Anti-BTNL8 mAbs inhibit activation of Vy4V61 TCR reporter cells.

Expression of the NF-AT-GFP reporter (A), CD69 (B) and TCRV51 (C) at the plasma membrane of JRT3-Vy4V51TCR reporter cells in coculture with HEK-BTNL3/BTNL8, in presence of chimeric anti-BTNL8 mAbs or corresponding isotype control were assessed by Flow cytometry. Dose-response of this inhibition as showed by IC50/EC50 assessed for each mAb and each readout.

Figure 5. Anti-BTNL8 mAbs inhibit activation of primary Vy2/3/4 cells. A. Expression of CD25 on Vy2/3/4+V51 + and Vy2/3/4-V51+ T cells assessed by FACS upon coculture of V51 T cells expanded from HD PBMCs with HEK-pLV-empty or H EK-BTN L3/BTN L8. B. Effect of anti-BTNL8 mAb on Vy2/3/4+V51+ cells activation upon coculture with H EK- BTN L3/BTNL8.

EXAMPLES:

Material and Methods

Generation of monoclonal antibodies (mAbs)

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 titer of BTNL8-specific polyclonal antibodies was determined via Luminex assay. Mice displaying the highest BTNL8-specific antibodies titer were euthanized. Splenic B cells were isolated via positive selection and underwent PEG- induced fusion to myeloma cells for hybridoma generation. Hybridomas were subcloned by limiting dilution and hybridoma supernatants underwent two rounds of screening for target specificity and their capacity to modulate V81 T cell function in vitro. Hybridoma supernatants were used as such, or mAbs were purified by affinity chromatography on protein-A resine (GE Healthcare) using an AKTA-Pure device (GE Healthcare).

Luminex assay

Magnetic COOH beads (Biorad) were conjugated to rhBTNL8-Fc protein (R&D) according to manufacturer’s instructions and beads were stored in storage buffer (Biorad) at -20°C until use. For titration of mouse sera, serial serum dilutions were made in Luminex assay buffer (Nanotools) starting at 1:50, by dilution steps 1:4; 100 pL bead suspension were mixed with 100 pL serum dilution and incubated for 1 hr at RT, after which beads were washed 3-times in washing buffer, incubated with 1 pg/mL biotinylated goat anti mouse IgG-Fc in Luminex assay buffer, and had 3 further washes in Luminex assay buffer. Finally, beads were incubated for 1 hr with 1 pg/mL streptavidin-PE in Luminex assay buffer, before 3 final washes in Luminex read buffer (Nanotools). Beads were resuspended in Luminex read buffer and data were acquired on a Luminex 100/200 system. For hit identification and affinity assessment, 30 pL supernatant were transferred into 96 well plates, and 90 pL Luminex assay buffer were added. One hundred microliters of bead suspension were mixed with 100 pL supernatant dilution and incubated for 16 hrs at RT, before proceeding to the protocol described above. For hit identification, those with the highest affinity for the target and the lowest affinity for an irrelevant control protein (Rank-Fc) were selected. FAB beads allowed estimation of antibody concentration; the midpoint of its binding curve corresponded to 100 pM antibody concentration. For affinity/Kd calculation, hybridoma supernatants underwent serial dilution in Luminex assay buffer starting at 40.000 pM, by dilution steps 1:4, and were analyzed as described above. Kd corresponds to midpoint of the corresponding binding curve. The ratio FAB (MFI) / target (MFI) allowed the prediction of antibody affinity.

Cell culture

Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of healthy donors (HD) provided by the local Blood Bank ( Etablissement Frangais du Sang (EFS)- Marseille-France) and isolated by centrifugation on density gradient (Eurobio). The following human cell lines were obtained from the American Type Culture Collection: HEK-293T (embryonic kidney), JRT3 T3.5 cells (Jurkat derivative devoid of apTCR), HL- 60 (myelogenous leukaemia) cells. HEK-293T cells and its derivatives were cultured in DMEM medium (Life Technologies) with 10% foetal calf serum (FCS). HL-60 cells, as well as JRT3.3 cells and its derivatives were cultured in RPMI 1640 medium supplemented with 10% FCS, 1% Na-Pyruvate, 1% L-glutamine (all from Life technologies). Hybridomas were cultured in DMEM/Ham’s F12 (1:1) (ThermoFisher Scientific), 4% FetalClone I (Hyclone), Chemically Defined Lipid Concentrate (1:250), 1% Glutamine, 1% sodium pyruvate and 100 pg/mL PenStrep (all from ThermoFisher Scientific). For collection of hybridoma supernatants, hybridomas were cultured for 4-5 days without Fetalclone.

Lentiviral transduction

For generation of lentiviral particles, HEK-293T cells were transfected with the following plasmid pMD2.G (encoding envelop glycoproteins VSV-G), psPAX2 (encoding HIV-1 derived proteins gag, pol, tat and Rev) and pLV lentiviral expression vector, which was either empty or encoding the indicated gene of interest, using TurboFect reagent (ThermoFisher) according to manufacturer’s instructions. Transfection medium was replaced by fresh OptiMEM™ (ThermoFisher) after 24h. Culture supernatant containing lentiviral particles was collected after 24h and 48h, and concentrated LentiX concentrator™ (Takara) following manufacturer’s instructions. For HEK-BTNL8 and HEK- BTNL8/BTNL3 transductants, optimized versions of wild-type human BTNL8 cDNA (NCBI Reference Sequence: NM_001040462.2), or wild-type human BTNL3 cDNA (NCBI Reference Sequence: NM_197975.2) with a c-Myc(EQKLISEEDL)-tag in N-terminal, were cloned into pLV vector using Hindlll/BamHI restriction sites. HEK-293T cells were seeded in 12-wells plates (2.5x10⁵ cells/well), and 25 pi of concentrated lentiviral particles were added to the culture. After 24h, cells were washed twice in complete medium, and put back in culture in their regular culture medium for 48h. Transductants were selected by addition of 1 pg/mL puromycin into the culture medium.

ELISA assay for cynomolgus BTNL8 ortholog cross-reactivity

Cynomolgus BTNL8 ortholog sequence (XP_005558887.1) was identified after BLAST search using human BTNL8 amino acid sequence, and its extracellular domain was cloned into pFUSE-hlgG1FC2 vector (InvivoGen) using EcoRI/EcoRV restriction sites. Recombinant cynoBTNL8-Fc fusion protein was produced by transfection of the resulting pFUSE-hlgG1 FC2-cynoBTNL8 plasmid into Expi293F™ cells with ExpiFectamine™ 293 (ThermoFisher) according to manufacturer’s instructions. The cell culture supernatant collected on day 6 was used for purification through an affinity purification column. The purified cynoBTNL8-Fc protein was analyzed by SDS-PAGE and Western blotting for molecular weight and purity measurements. cynoBTNL8-Fc protein concentration was determined by Bradford assay with BSA as a standard. For ELISA, cyno BTNL8-Fc protein (1 pg/mL in 1X PBS) was coated overnight at 4°C. After 3 washes in PBS, plates were saturated with BSA 2% v/v in PBS for 1 h at room temperature, then saturating buffer was discarded. Anti-BTNL8 hybridoma supernatants were diluted ½ in PBS BSA 2%, and 100 pL were added 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, 1:10000 dilution in PBS BSA 2%) and incubation for 1 h at room temperature. Then, all wells were washed 3 times in PBS and 1-step ABTS solution (ThermoFisher) was added for binding revelation, as assessed by absorbance at 405 nm in a Spark spectrometer (Tecan).

Generation of Vy4V51TCR reporter cells

A bi-cistronic construct containing a human Vy4 TCR chain and a human V51 TCR chain separated by a P2A sequence (IC202-g4_P2A_d1: [SEQ ID NO:84]) was designed, synthesized and cloned into pLV vector using BamHI/Sall restriction sites. The NFAT- EF1a-eGFP reporter lentiviral vector was generated using the pLV-Blasticidin- NFAT- EF1a-eGFP vector (Custom DNA Constructs). JRT3 T3.5 cells were transduced concomitantly with lentiviral particles encoding the NFAT-EF1a-eGFP reporter and either the IC202-g4_P2A_d1 construct or an empty pLV, as described above. JRT3 T3.5 transductants were selected in culture medium containing puromycin (1 pg/mL) and blasticidin (10 pg/mL). p24 negative status of the transductants was confirmed using Lenti X ™ p24 Rapid Titer kit (Takara). JRT3 transductants with the highest plasma membrane expression of Vy4V51TCR were sorted by flow cytometry using anti-V61-PE-Vio770 (Miltenyi) in a BD-Aria II Cell Sorter (Becton Dickinson). JRT3 T3.5 transductants bearing Vy4V51TCR were named JRT3-Vg4Vd1-202 reporter cells in all experiments.

Vy4V51TCR reporter cells assay

JRT3-Vg4Vd 1-202 reporter cells were co-cultured with HEK-BTNL8/BTNL3 cells (ratio 1:1, 5x10⁴ cells each; culture volume 100 pL) with or without 50 pL anti-BTNL8 or hybridoma culture medium (HCM) and incubated for 16 hrs at 37°C 5% CO2. Hybridoma culture medium (HCM) was added to the co-culture as negative control (reference). JRT3- Vg4Vd 1-202 reporter cells alone (5x10⁴ cells) were incubated with PMA (20 ng/ml_)/lonomycin (1 pg/mL) (Sigma), or anti-CD3 (clone OKT3) as positive control; or with lgG2b (isotype control for OKT3). At the end of the incubation time, cells were spun down and pellets were resuspended in a staining mix containing anti-CD69-APC (BD Biosciences), anti-V61-PE-Vio770 (Miltenyi) and Live/Dead Aqua fluorescent dye (Life Technologies). Median fluorescence intensity (MFI) was assessed on an Intellycyt l-QUE cytometer and analyzed with a FlowJo software. The variations (D%) of CD69 and TCRV51 expression at the plasma membrane (MFI), and of NFAT-eGFP reporter (%GFP+ cells) relative to the reference were calculated for each sample. Anti-BTNL8 antagonists of Vy4V51 activation were those with the lowest CD69 and NF-AT A% below the corresponding median D% of all samples AND the highest TCRV51 D% above the median A% of all samples.

Expansion of V81 T cells

Panyd T cells were isolated from fresh PBMCs by negative selection using EasySep™ Human gamma/delta T cells isolation kit (Stemcell Technologies) following manufacturer’s instructions. Purified gdT cells were cultured at 37°C, 5% CO2 in Roswell Park Memorial Institute medium 1640 (RPMI) supplemented with 10% human serum in presence of mitomycin C-treated autologous PBMCs, human cytokines (rhlL-4, rhlL-1 p, rhlL-21 and rhlFN-g, all from Miltenyi) and soluble anti-CD3 (clone OKT3, Thermofisher). Mitomycin C- treated autologous PBMCs were added to the culture as feeder cells (ratio 1:1) on day 0 and were renewed after one week. Every 4 to 5 days, culture medium was replaced by fresh medium supplemented with cytokines and anti-CD3 mAb. rhlL-4 was replaced by rhlL-15 after one week. Frequency of V51 T cells among expanded gdT cells was monitored by flow cytometry on day 0, and weekly during 3 weeks. Expanded V31 T cells were then frozen at -150°C in fetal bovine serum (FBS) supplemented with 10% Dimethyl Sulfoxyde (DMSO) for further analysis.

Degranulation assay on V81-T cells

Frozen expanded V31 T cells from healthy donors were pre-incubated overnight with human cytokines (rhlL-2 100UI/ml_ and rhlL-15 10 ng/mL). The next day, expanded V31 T cells were co-cultured ratio 1:1 with BTNL8-expressing human leukemia cell line (HL-60) as target cells, in presence of anti-CD107a and anti-CD107b antibodies and Golgistop, for 4 hours. Fifty microliters of anti-BTNL8 hybridoma supernatant or control medium were added to the co-culture during these 4 hours incubation. Anti-V61 TCR antibody was used as positive control for V61 T cells activation. After 4 hours of incubation, cells were stained extracellularly with a cocktail of fluorochrome-labelled mAbs (anti-CD3, anti-PanybTCR, anti-V61 TCR and Live/dead) and were analyzed by flow cytometry (Cytoflex LX, Beckman Coulter). The degranulation was determined by the percentage of CD107ab among V31 T cells (defined as CD3+ Pany3TCR+V31+).

Flow cytometry

PBMCs, purified V81-T cells or cell lines were incubated with specified mAbs before analysis on a CytoFlex LX or CytoFlex S (Beckman Coulter), or an l-QUE (Sartorius) cytometer, using FlowJo 10.5.3 software (FlowJo). Antibodies used for V81-T cell degranulation assay were anti-CD107a-FITC (BD Biosciences), anti-CD107b-FITC (BD Biosciences), anti-CD3-Alexa Fluor 700 (Biolegend), anti-PanybTCR-PE (Miltenyi), anti- V81TCR PE-Vio770 (Miltenyi) Live/dead near IR (Thermofisher). All immune stainings were performed using 10 pg/mL of purified mAbs, in presence of FcR Block reagent (Miltenyi), goat anti-mouse-PE 1:100 (Jackson Immunoresearch), and live/dead near IR (Thermofisher). Biotinilated anti-myc mAb (ThermoFisher) was used for detection of myc- tagged BTNL3 in HEK-BTNL3 cells.

Statistics

For V81-T cell degranulation, results are expressed as median ± SEM. All analyses were performed using GraphPad Prism 7.04 software (GraphPad).

Generation of recombinant chimeric anti-BTNL8 mAbs

VH and VL sequences of the reference anti-BTNL8 mAbs were synthesized in vitro and amplified by PCR using PrimeSTAR Max DNA Polymerase (Takara). PCR products were cloned in heavy chain and kappa light chain expression vectors (MI-mAbs) using In Fusion system (Clontech), and plasmids were transformed into Stellar competent cells (Clontech). Vector sequencing (MWG Eurofins) was performed in order to validate parental anti-BTNL8 sequences, before large scale (maxi) preparation of plasmid for further transfection. Vectors encoding matched light and heavy chains for each anti- BTNL8 clone were transiently transfected in HEK293-Expi cells (Thermofisher) 2.9x10⁶ of cells/mL) with a ratio heavy chain/light chain 1:1.2, and medium was renewed after 18h. Seven days after transfection, culture supernatants were harvested for mAb purification. Affinity purification of antibodies was performed with Protein A Sepharose Fast Flow (GE Healthcare), overnight at 4°C. Binding buffer was 0.5 M Glycine, 3M NaCI, pH8.9. Elution was performed with the following buffer: 0.1 M Citrate pH3. Samples were neutralized right after elution with 1M Tris-HCI, pH9 (10% v/v). Finally, chimeric anti-BTNL8 mAbs were dialyzed into PBS 1X and filtered through 0.22 mM filters (Millex GV hydrophilic PVDF, Millipore). Chimeric anti-BTNL8 mAb concentration was determined in a Nanodrop 2000 Spectrophotometer (ThermoScientific) taking into account the extinction coefficient of the antibodies. Purity, as defined by the fraction of mAb monomers, was determined by UPLC-SEC using an Acquity UPLC-HClass Bio (Waters), with an Acquity UPLC Protein- BEH-200A, 1.7 pm 4.6x 50 mm column (Waters). Antibody mass was determined in a Xevo G2-S Q-Tof mass spectrophotometer (Waters) using a reversed phase column (PLRP-S 4000 A, 5 pm, 50 x 2.1 mm (Agilent technologies). BTNL8 mAb expression on whole blood, epithelial cells and HEK cells

Fresh normal adjacent tissues of colorectal adenocarcinoma patients were obtained via BiolVT, and epithelial cells 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 local blood bank (EFS). CaCo-2 (colon adenocarcinoma) cells were purchased from ECACC Collection and were cultured in DMEM + 20% FBS + 1mM NaPy.

Staining of whole blood, normal epithelial cells, CaCo-2 and HEK cells was performed using purified mAbs (mouse or chimeric mAb) in presence of FcR Block reagent (Miltenyi), goat anti-mouse or anti human PE 1:100 (Jackson Immunoresearch), and live/dead (Thermofisher). For whole blood, a cocktail of fluorochrome-labelled 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 respectively in whole blood, while anti-Epcam (Biolegend) mAb was used to identify epithelial cells in tissues-isolated cells. For whole blood staining, red blood cells were depleted before acquisition using Cal-Lyse Lysing Solution (Thermofisher). The analysis was done on a CytoFlex LX or CytoFlex S (Beckman Coulter) or an l-QUE (Sartorius) cytometer, using FlowJo software.

Coculture of primary V51 T cells with HEK cells

Fresh or frozen V51 T cells expanded from healthy donors PBMCs were pre-incubated overnight with rhlL-2 (50UI/mL), and HEK cells (pLV-empty or BTNL3/BTNL8) were plated to form a monolayer. The next day, expanded V51 T-cells were co-cultured at ratio 1:1 with HEK-pLV-empty or HEK-BTNL8 in presence of 10 pg/mL of anti-BTNL8 mAb or corresponding isotype control for 24 hours. At the end of the incubation time, cells were stained extracellularly with a cocktail of fluorochrome-labelled mAbs (anti-CD3, anti- Pany6TCR, anti-V61 TCR and, anti Vy2/3/4 TCR (kindly provided by Pr. Dieter Kabelitz), anti CD25 and Live/dead) and were analyzed by flow cytometry (Cytoflex LX, Beckman Coulter).

Binding Affinity by Biolayer Interferometry Assay

The Biolayer Interferometry Assay (BLI) was performed as follows: Anti-human IgG Fc Capture Biosensors (AHC; Fortebio) first hydrated with 0.2 ml kinetic buffer (PBS pH 7.4, 0.02% Tween20 and 0.1% BSA) for 10 min and then loaded with hBTNL8 (2 pg/mL). The association of antibody with various concentrations of hBTNL8 (40, 20, 10, 5, 2.5 and 1.25 nM) was monitored for 150 s, and the dissociation was followed for typically 300 s in KB. All runs (including loading, equilibration, association/dipping of sensors into analyte, dissociation, and regeneration) were performed in black 96-well plates (Greiner) at 30 °C with shaking 1000 rpm using an OctetRed96 (ForteBio). A control with an irrelevant antibody was used to assess the absence of non-specific binding of the target protein. Fitting of the data and constant measurements were performed with the Octet Red system software (version 11.1) using the 1:1 model. The equation used to fit the association is an integration of a differential equation showing that the rate of association is a function of the decreasing concentration of unbound ligand molecules as analyte binding occurs:

Y = Y₀ + A(1 - e^~kobs*^t) where Y = level of binding, Yo= binding at start of association, A is an asymptote and t = time. k₀bs is the observed rate constant.

The equation used to fit dissociation is:

Y = Yo + Ae-^ where Yo is binding at start of dissociation, and k_d is the dissociation rate constant. By solving the above equations for kobs and kd, the association rate constant ka can then be calculated with the equation:

Finally, the affinity constant K_D is calculated using k_a and k_d:

^Kd=? ka

Epitope binning by Biolayer Interferometry Assay

Epitope binning is performed using BLI, as described above. Test antibody is immobilized on the sensor. Recombinant human BTNL8 is then presented at 40 nM and, when association reaches saturation, a second (competitor) antibody is presented at 40 nM. If the competitor antibody binds overlapping epitopes with the test antibody, no additional binding curve will be observed. If the competitor antibody binds to a non-overlapping epitope, a second binding curve will be observed. The test antibody is presented as free ligand to confirm lack of binding with overlapping epitopes and use the signal as reference. Binning data were analyzed using Octet Data Analysis HT 11.1 using epitope bin operation. RESULTS

Identification of the reference anti-BTNL8 hybridomas

The reference anti-BTNL8 antibodies were identified as follows:

Mice were immunized with BTNL8-Fc antigen and splenocytes from mice presenting with the highest titer of BTNL8-specific sera were collected and fused with myeloma to obtain hybridomas. Hybridoma culture supernatants displaying the highest affinity for BTNL8 (n=320), as assessed on Luminex assay, underwent a first round of screening based on their ability to bind human BTNL8 in cellulo. As shown in figure 1C, 310/320 anti-BTNL8 hybridoma supernatants were able to stain HEK-BTNL8 and all of them stained HEK- BTNL8/BTNL3 transductants, but not wild-type HEK293T cells or HEK-BTNL3 transductants, as assessed on flow cytometry. In order to identify clones that presented with immunomodulatory properties on V51 T cells, anti-BTNL8 hybridoma supernatants were tested for their ability to modulate activation of Vy4V51TCR reporter cells, and then to modulate the cytotoxicity of expanded V51 T cell against BTNL8-expressing cancer cells. Selected clones from this first round screening underwent subcloning, which resulted in 81 subclones of anti-BTNL8 hybridomas that were retested for the same criteria.

BTNL8 targeting with anti-BTNL8 mAbs allows modulation of BTNL8/BTNL3- induced activation through Vy4V51TCR.

JRT3-T3.5 T cells, which are Jurkat T cell line derivative devoid of TCRap, were transduced to express a colon-derived Vy4V51TCR and a GFP reporter transgene under the control of the NF-AT promoter. These cells were called JRT3-Vg4Vd 1-202 reporter cells. In parallel, HEK-293 cells that stably express BTNL8 and BTNL3 (HEK-L8/L3) were generated. Given the ability of the BTNL8/BTNL3 dimer to trigger activation through the Vy4V51TCR in gut V51 T cells (Di Marco Barros et al. Cell, 2016), we assessed whether anti-BTNL8 hybridoma supernatants could modulate the activation induced by co-culturing JRT3-Vg4Vd 1-202 reporter cells with HEK-BTNL8/BTNL3 cells, compared to an irrelevant hybridoma supernatant (Figure 2A and B). Forty-seven anti-BTNL8 hybridoma supernatants were found to modulate BTNL8/BTNL3-induced JRT3-Vg4Vd 1-202 reporter activation out of 320 anti-BTNL8 hybridoma supernatants tested, in contrast to hybridoma culture medium and irrelevant control hybridoma supernatant (Figure 2B). In particular, thirty-five anti-BTNL8 hybridoma supernatants induced upregulation of TCRV51 expression and downregulation of NFAT-GFP reporter and CD69 (Figure 2C and Table 3). Forty-seven clones including these 35 anti-BTNL8 hybridoma supernatants were selected for next step of screening and sub-cloning.

Table 3: Anti-BTNL8 hybridoma supernatants selected on BTNL8/BTNL3-JRT3- Vg4Vd1-202 reporter assay. Variations (D) of NFAT-GFP reporter, CD69 and TCRV51 expressions compared to co-cultures of JRT3-Vg4Vd 1-202 reporter cells with HEK- BTNL8/BTNL3 in presence of hybridoma culture medium.

Anti-BTNL8 antibodies induce V51 T cell degranulation against BTNL8-expressing cancer cells

Although BTNL8/BTNL3-mediated activation through the Vy4V51TCR has been described in the literature (Melandri et al. Nat Immunol, 2018), its outcome in V51 T cells is yet unknown. We hypothesized that BTNL8/BTNL3-mediated Vy4V51TCR activation could be involved in V51 T cell cytotoxicity. In order to test this hypothesis, purified V81 T cells were expanded from PBMCs of healthy donors and co-cultured with HL-60 leukemia cells, which express BTNL8 at the plasma membrane (Figure 3A), as target cells, with or without anti-BTNL8 hybridoma supernatants. Interestingly, we found that expanded V51 T cell degranulation against HL-60 leukemia cells was enhanced 2-3 folds by anti-V51 TCR antibody (Figure 3B). More importantly, V51 T cell degranulation was modulated by 23 out of 47 anti-BTNL8 hybridoma supernatants initially selected for modulating activity of reporter assay. Indeed, the addition of 23 anti-BTNL8 hybridoma supernatants led to an inhibition of the cytolytic function of expanded V81 T cells, as measured by the percentage of CD107+ degranulating cells, compared to co-cultures with HL-60 cells alone, or in presence of control hybridoma culture medium. PMA/ionomycin treatment of V81 T cells lead to maximum induction of their cytolytic function without addition of HL-60 cells, as expected.

The fraction of CD107+ cells induced by anti-BTNL8 hybridoma supernatant ranged from 7.98% to 46.4% CD107+ cells, compared to co-cultures with control hybridoma culture medium (17.2%). In co-cultures of expanded V81 T cells and BTNL8-expressing HL-60 cells, anti-BTNL8 supernatants resulted in 1.44 to 2-fold inhibition of expanded V81 T cell degranulation compared to the same co-cultures in presence of control hybridoma culture medium.

In order to confirm the V51 T cell antagonist activity found for some anti-BTNL8 hybridoma supernatants during primary screening, the initial hits were subcloned and anti-BTNL8 mAbs were purified from hybridoma supernatants of the resulting subclones. Purified anti- BTNL8 hybridoma supernatants were screened for their ability to modulate V51 T cell degranulation against HL-60 cells. Twenty-four purified anti-BTNL8 mAbs were found to inhibit degranulation of V51 T cells against HL-60 cells by ³15% reduction compared to their control isotype mAb. Table 4 show the fraction of degranulating (CD107ab+) V51 cells obtained in presence of these 24 antagonist anti-BTNL8 mAbs tested for 3 different healthy donors. Table 4: V51 T cell degranulation against HL-60 cells in presence of V51 antagonist anti-BTNL8 mAbs

The reference V51 antagonist anti-BTNL8 mAbs recognize the BTNL8 but not BTNL3 in cellulo In order to establish that the V51 antagonists anti-BTNL8 antibodies identified recognize BTNL8 but not its molecular partner BTNL3, HEK-293T cells were transduced with lentiviral vectors encoding BTNL8, or BTNL3 or both. Staining of HEK-293T WT, HEK- BTNL8, HEK-BTNL3 or HEL-BTNL8/BTNL3 with the selected 24 anti-BTNL8 Abs was only detected in HEK-BTNL8 and HEK-BTNL8/BTNL3, but not HEK-293T WT or HEK- BTNL3 cells alone in all clones but mAb34 and mAb47 (data not shown). mAb34 displayed weak staining in HEK-293T WT and HEK-BTNL3. mAb47 displayed staining in HEK-BTNL3, indicating cross-reactivity with BTNL3.

After sequencing of these anti-BTNL8 mAbs, we provide the sequences of 5 reference Vy1 antagonist antibodies, which show Vy1 antagonist activity and bind to HEK-BTNL8, HEK BTNL8/BTNL3, but not HEK-BTNL3, in table 10.

Affinity of the reference V51 antagonist anti-BTNL8 antibodies mAb for BTNL8

Affinity of the reference anti-BTNL8 antibodies for recombinant human BTNL8-Fc protein was assessed by testing their corresponding hybridoma supernatants using Luminex. Data are summarized in Table 5. Table 5: Luminex based assessment of the affinity of the reference anti-BTNL8 mAbs

Cross-reactivity of the reference anti-BTNL8 mAbs for cynomolgus BTNL8 ortholog

BTNL8 orthologs are present in most non-human primates, including cynomolgus ( Macaca fascicularis). In order to determine the cross-reactivity of the reference anti-

BTNL8 mAbs with cynomolgus BTNL8 ortholog (cynoBTNL8; NCBI ref. XP_005558887.1 , 88% identity to human BTNL8), we generated a recombinant Fc-fusion protein containing the ectodomain of cynoBTNL8 (cynoBTNL8-Fc) and we performed ELISA assay for assessing the binding of hybridoma supernatants from the reference anti-BTNL8 mAbs to this protein. As shown in Table 6, the reference V51 antagonistic anti-BTNL8 mAbs were able to bind cynoBTNL8-Fc in our ELISA assay (optical density (OD) at 405 nm > 0.2, threshold set over hybridoma culture medium (HCM) absorbance), in contrast to an irrelevant control mAb (anti-His). Three out of 18 V51 antagonistic anti-BTNL8 mAbs (mAb1, mAb9, mAb34) did not bind to cynoBTNL8-Fc, meaning no cross-reactivity to cynomolgus BTNL8 ortholog (data not shown). As expected, anti-Fc mAb, used as positive control for cynoBTNL8-Fc binding, yielded a strong positive signal (OD405 nm= 1.293). Table 6: ELISA assessment of binding of the reference V51 antagonistic anti-BTNL8 hybridoma supernatants to cynomolgus BTNL8 ortholog.

Affinity, cross-reactivity, and epitope binning of reference anti-BTNL8 mAb Affinity of reference mAb on chimeric format was first assessed for their binding in cellulo by flow cytometry. In cellulo, all reference anti-BTNL8 mAb, but not mAbX3, bind BTNL8 and BTNL3/BTNL8 heterodimer on HEK cells with high affinity (< 0.5 pg/ml = 3.3 nM) (data not shown) and the corresponding EC50 are presented in the table 7 below: Table 7: Binding of 4 selected chimeric anti-BTNL8 mAbs on HEK-BTNL8 cells determined by flow cytometry

Similar results were obtained for binding to recombinant BTNL8 protein determined by bio-layer interferometry (BLI) and the affinity constants (KD, k_d, k_a) of the selected anti- BTNL8 chimeric mAbs are presented in the table below:

Table 8: Kinetic parameters of chimeric antibody binding to human BTNL8 protein as determined by BioLayer Interferometry (BLI)

mAbX1, mAbX2, mAbX4 and mAbX5 bind BTNL8 with sub-nanomolar affinities and mAbX3 was found not to bind BTNL8.

In addition, mAbX1, mAbX2, mAbX4 and AbX5 bind to recombinant cynoBTNL8-Fc protein, indicating a cross-reactivity of the reference anti-BTNL8 mAb with cynomolgus BTNL8 ortholog (data not shown).

Then, we investigated whether the reference mAbs recognized the same BTNL8 epitope region. Therefore, octet-based binning experiments were performed where the 4 binder mAbs competed for BTNL8 binding using “classical sandwich” setting. The mAbX1 did not block binding of the other 3 mAbs to BTNL8, but mAbX2, mAbX4 and mAbX5 blocked each other’s binding, indicating that these mAbs bind to overlapping epitope region on BTNL8, and that mAbX1 binds a different epitope as shown in the table below:

Table 9: Matrix of Pearson correlation coefficients between saturating mAbs (mAbX1, columns) and the blocking mAbs (mAbX2, rows). The strongest correlations appear in black, and weak correlations appear in white. Antibody self-binding pairs values appeared bold.

Reference anti-BTNL8 mAb binds to BTNL8 on normal and tumor epithelial cells from human gut tissue (data not shown)

According to the Human Protein Atlas, BTNL8 mRNA is detected at high level on enterocytes, neutrophils and CaCo-2 cells. We determined BTNL8 expression at the plasma membrane of primary normal and tumor gut cells by using our reference anti- BTNL8 mAbs. mAbX1, mAbX2, mAbX4 and mAbX5 stained CaCo-2 cells with variable intensities. Strong staining was also observed on primary epithelial cells (Epcam+ cells) from normal tissues using mAbXl. No binding of these reference anti-BTNL8 mAbs was detected on immune cells from peripheral blood, including neutrophils.

Reference anti-BTNL8 mAbs strongly inhibit Vy4V51 T cell activation induced by BTNL3/BTNL8 dimer

Previous data obtained with hybridoma supernatants showed an antagonist effect of the reference BTNL8 mAbs on the activation of Vy4V51TCR reporter cells as showed by increased TCRV51 expression at the plasma membrane and inhibition of NFAT-GFP reporter and CD69 expression. This antagonist activity is also confirmed when using the reference chimeric anti-BTNL8 mAbs (Figure 4). Antagonist activity of chimeric anti- BTNL8 mAbs is potent, as evidenced by IC50/EC50 <0.5 ug/mL (<3.3 nM) for all readouts tested (Figure 4).

Reference anti-BTNL8 mAbs are able to inhibit primary Vy4V51 T cells activation induced by BTNL3/BTNL8 dimer

We sought to determine if the reference anti-BTNL8 mAb could modulate primary Vd1 T cell activation. To address this question, we first tested the ability of BTNL3/BTNL8 dimer to activate primary Vd1 T cells subsets expanded from healthy PBMC.

Coculture with HEK-BTNL3/L8 cells induced a consistent activation of Vy2/3/4+ V51 T cells compared to HEK-pLV empty cells in all donors tested (Figure 5A). This activation was inhibited by mAbXl, mAbX2, mAbX4 and mAbX5 but not mAbX3 (Figure 5B). No relevant effect was observed on Vy2/3/4- V51 T cells subsets (data not shown).

The weaker antagonist activity of reference anti-BTNL8 mAb in this assay compared to the Vy4V51TCR reporter assay is probably due to the heterogeneity of the proportion of Vy4-bearing cells within primary Vy2/3/4+ V51 T cells from different samples, as detected by anti Vy2/3/4 mAb. Table 10: Brief description of useful amino acid and nucleotide sequences for practicing the invention (CDRs regions in VH/VL sequences are highlighted)

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 into the present disclosure.

Claims

1. An antibody having specificity for human BTNL8 (BTNL8), characterized in that it has one or more of the following properties: i. it binds to human BTNL8/BTNL3 dimer as expressed in a cell line, for example HEK-293T cells, and/or, ii. it does not bind to a cell line expressing BTNL3 but not BTNL8, for example HEK-293T cell lines expressing BTNL3.

2. An antibody having specificity for human BTNL8 (BTNL8), characterized in that it binds to human BTNL8-Fc with a K_D below 10 pM, for example as measured by Luminex assay.

3. The anti-BTNL8 antibody according to Claim 1 or 2, characterized in that it has at least one of the following properties: i. it inhibits the activation of T cells bearing a V51 TCR (V51 T cells), ii. it inhibits the cytolytic function of activated V51 T cells, and/or, iii. it inhibits the production of cytokines by activated V51 T cells.

4. The anti-BTNL8 antibody of any one of Claims 1 to 3, characterized in that it has one or more of the following properties: i. it inhibits the activation of Vy4V61 TCR bearing T cells, typically as determined by Vy4V51 TCR reporter cell assay; and/or, ii. it inhibits the cytolytic function of activated V61 T cells, typically it inhibits the degranulation of V51 T cells against HL-60 cells, as determined in an in vitro degranulation cellular assay;

5. The anti-BTNL8 antibody of any one of Claims 1 to 4, characterized in that it inhibits the activation of Vy4V61 TCR bearing T cells with a with an IC50 below 1.1 nM as measured with CD69 expression at the plasma membrane as a readout.

6. The anti-BTNL8 antibody of any one of Claims 1-5, which cross-reacts with cynomolgus BTNL8.

7. The anti-BTNL8 antibody of any one of Claims 1 to 6, which competes for binding to BTNL8 with at least one of the following reference murine antibodies: i. The 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; ii. The 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; iii. The 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; or, iv. The 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.

8. The anti-BTNL8 antibody of any one of Claims 1 to 7, comprising either, i. the H-CDR1 , H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb X1 of SEQ ID NOs:11-16 respectively; ii. the H-CDR1 , H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb X2 of SEQ ID NOs: 17-22 respectively; iii. the H-CDR1 , H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb X4 of SEQ ID NOs:29-34 respectively; or, iv. the H-CDR1 , H-CDR2, HCDR3, L-CDR1, L-CDR2 and L-CDR3 of the mAb X5 of SEQ ID NOs:35-40 respectively.

9. The anti-BTNL8 antibody of any one of Claims 1 to 8, which is an antibody comprising either, i. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:1 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:2; ii. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:3 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:4; iii. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:7 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:8; or, iv. a heavy chain wherein the VH region has at least 90% identity with SEQ ID NO:9 and a light chain wherein the VL region has at least 90% identity with SEQ ID NO:10.

10. The anti-BTNL8 antibody of any one of Claims 1 to 9, which is a human, chimeric or humanized antibody.

11. A nucleic acid molecule which encodes a heavy chain and/or a light chain of the anti- BTNL8 antibody of any one of Claims 10.

12. A host cell comprising the nucleic acid of claim 11.

13. The anti-BTNL8 antibody of any one of Claims 1-10, for use as a medicament.

14. The anti-BTNL8 antibody of any one of Claims 1-10, for use in treating inflammatory disorders, for example, inflammation in the gut of a subject.

15. The anti-BTNL8 antibody of any one of Claims 1-10, for use in treating inflammatory disorders selected from inflammatory bowel disease (IBD), in particular Crohn’s disease, celiac disease or ulcerative colitis.

16. A pharmaceutical composition comprising the anti-BTNL8 antibody of any one of Claims 1-10, and at least a pharmaceutically acceptable carrier.