OA19665A

OA19665A - Antibodies and polypeptides directed against CD127.

Info

Publication number: OA19665A
Application number: OA1201900214
Authority: OA
Inventors: Nicolas Poirier; Caroline Mary; Bernard Vanhove; Virginie Thepenier
Original assignee: Ose Immunotherapeutics
Priority date: 2016-12-09
Filing date: 2017-12-07
Publication date: 2020-12-31

Abstract

The invention is in the field of antibodies useful in therapeutic and diagnostics applications targeting CD127, the alpha chain of the IL7 receptor, and provides in particular humanized monoclonal antibodies against CD127, particularly human CD127, therapeutic uses thereof, and diagnostics applications.

Description

ANTIBODIES AND POLYPEPTIDES DIRECTED AGAINST CD127

[1] The invention is in the field of polypeptides or antibodies useful in therapeutic and diagnostics applications targeting CD 127, the alpha chain of the 1L7 receptor, and provides in particular humanized monoclonal antibodies against CD127, particularly human CD127, therapeutic uses thereof, and diagnostics applications.

Brief description of the invention

[2] The alpha chain (or subunit) of the receptor for interleukin7 (IL-7), is designated CD 127 or p90 IL-7R or IL-7Ralpha or IL-7Ra (sometimes also noted IL-7Ra). In a particular aspect, the antibodies, particularly monoclonal antibodies, provided herein are directed against the alpha chain of the receptor for human IL-7 expressed on human cells. In a particular aspect, the antibodies provided herein hâve antagonist properties for IL-7-IL-7R interaction, may présent cytotoxic activity against CD 127 positive cells but do not increase the maturation of dendritic cells (DCs) induced by TSLP. In particular, the antibodies provided herein recognize a human CD127 epitope comprising sequences from the 2b site of CD 127, in particular the epitope comprises human CD127 sequences of domain DI and of the 2b site of CD 127, in particular the epitope comprises at least one sequence from DI and at least one sequence from the 2b site, preferably from the third beta sheet of the 2b site of CD 127. Alternatively, or in addition, in a particularaspect, the antibodies provided herein do not induce the internalization of CD127 and/or inhibit the IL7-induced internalization of CD127. In a particular aspect, the antibodies provided herein are humanized and comprise at least 80 % and preferably at least 84 %, or at least 85 % of human residues. In a particular aspect, the antibodies provided herein hâve a fast effect on effector memory T cells, a long-lasting effect on effector memory T cells, or, preferably, a fast and long-lasting effect on effector memory T cells. In a particular aspect, the antibodies provided herein allow for efficient production. In a particular aspect, the expression levels of CD127, IL-7 and/or TSLP may be measured to assess likelihood of response, in particular using the anti-CD127 antibodies provided herein. In addition to antibodies, provided herein, in particular as tools for the production of antibodies and/or for uses similar to said antibodies, are polypeptides, in particular antibody light chain variable domains or antibody light chains, antigen-binding fragments and antibody mimetic molécules from such antibodies and polynucleotides.

[3] The inventors hâve sought to obtain improved humanized antibodies when compared to the N13B2-hl, N13B2-h2 and N13B2-h3 antibodies disclosed in WO 2015/189302. While it is known that some residues in the variable domain framework sequences, including residues in the Vernier zone, canonical residues, residues at the VH/VL interface, etc, are critical in the structure of an antibody and should not be mutated in order to preserve the biochemical and biological activity of an antibody, the inventors hâve suprisingly discovered that some mutations within the light chain variable domain framework sequence of N13B2, including some of such critical residues, allow increasing human residue content (higher than N13B2-h3 and up to 86,3%) and improved production (up to 4 times higher thanN13B2-h3) while preserving ail the functional features.

[4] These mutations consist of 16 aminoacids substitutions within the light chain variable domain framework sequence ofN13B2 and in particular additionnaly in the substitution of the valine residue at position 48 by a leucine residue (V48L) and/or in the substitution of the phenylalanine residue at position 87 by a tyrosine residue (F87Y). These positions, and any amino acid positions with antibody chains provided herein, unless otherwise stated, are provided using Kabat numbering. Provided herein are the improved antibody light chain variable domains consisting of the following sequences:

- SEQ ID No: 9 (including said 16 aminoacids substitutions - said antibody light chain variable domain being hereinafter designated N13B2-hVL3);

- SEQ ID No: 10 (including said 16 aminoacids substitutions and additionnaly with mutation V48L - said antibody light chain variable domain being hereinafter designated N13B2-hVL4);

- SEQ ID No: 11 (including said 16 aminoacids substitutions and additionaly with mutation F87Y - said antibody light chain variable domain being hereinafter designated N13B2-hVL5); and

- SEQ ID No: 12 (including said 16 aminoacids substitutions and additionnaly with both mutations V48L and F87Y - said antibody light chain variable domain being hereinafter designated N13B2-hVL6).

[5] Thus, the antibody light chain variable domain designated herein N 13B2-hVL3, comprises the CDRs of N13B2-h3, said 16 aminoacids substitutions and the original residues of N13B2 at critical positions 48 and 87 (i.e., respectively, V and F) within human framework sequences and has the sequence of SEQ ID No: 9. The light chain variable domain of the preferred improved antibody provided herein, designated N13B2-hVL6, comprising in addition both mutations V48L and F87Y, consists of the sequence set forth in SEQ ID No: 12.

[6] The skilled person would not hâve expected that said mutations, particularly at such positions in the frameworks of the light chain variable domain would leave the afïïnity of the antibody with CD 127 unaffected: V48 is a canonical residue and F87 is located at the VH/VL interface; the mutation of such residues, as is known to the skilled person and discussed in e.g. Clark, 2014, is expected to affect affmity and/or stabiliy of the antibody. As used herein, the term canonical residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al., J. Mol. Biol. 196:901- 907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992). According to Chothia et al., critical portions ofthe CDRs of many antibodies hâve nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. Each canonical structure spécifiés primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop. It was not foreseeable that the introduction of the disclosed mutations at both positions would resuit in improved production, while still preserving binding features and advantageously other functional features of the antibodies.

[7] Accordingly, provided herein is a polypeptide useful in particular for the production of such antibodies, in particular an antibody light chain variable domain or an antibody light chain, consisting of a sequence, in particular of up to 250 amino acids, comprising or consisting of the sequence set forth in SEQ ID NO : 9; SEQ ID NO : 10; or SEQ ID NO : 11; or SEQ ID NO : 12, or an antigen-binding fragment or antibody mimetic molécule thereof as defined hereinafter. In a particular aspect, the invention relates to antibodies or antigen-binding fragment thereof, provided herein, particularly humanized monoclonal antibodies, specifically bind to CD 127, particularly to human CD 127, and comprise:

- an antibody light chain comprising or an antibody light chain variable domain consisting of a sequence selected from the group consisting of SEQ ID No: 9; SEQ ID No: 10; SEQ ID No: 11; SEQ ID No: 12; in particular SEQ ID No: 12; and

- an antibody heavy chain variable domain comprising the three CDRs consisting of the sequences set forth in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, in particular an antibody heavy chain variable domain consisting of the sequence set forth in SEQ ID No: 7.

In a preferred embodiment, the invention relates to antibodies or antigen-binding fragments thereof, provided herein, particularly humanized monoclonal antibodies, that specifically bind to CD 127, particularly to human CD 127, and comprise:

- a light chain variable domain consisting of a sequence selected from the group consisting of SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, particularly SEQ ID No: 12;and

- a heavy chain variable domain comprising the three CDRs consisting of the sequences set forth in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, in particular a heavy chain variable domain consisting of the sequence set forth in SEQ ID No: 7.

[8] In a particular aspect, the antibody provided herein comprises a light chain with a variable domain as above and a constant domain consisting of a sequence selected among SEQ ID No: 27 or 28, preferably SEQ ID No:27 and a heavy chain with a variable domain as above and a constant domain consisting of the sequence set forth in SEQ ID No:26.

[9] Also provided herein is an isolated polypeptide consisting of a sequence of up to 250 amino acids, in particular of up to 217, of up to 214, of up to 211, more particularly of up to 200, of up to 175, of up to 150, of up to 135, of up to 120, of up to 107 and even more particularly of up to 100, of up to 90, of up to 80, of up to 74, of up to 70, of up to 60 amino acids, wherein said sequence comprises or consist of a sequence selected from the group consisting of:

- SEQ IDNo: 9 (CDRs ofN13B2-h3 within human framework sequences);

- SEQ ID No: 10 (CDRs of N13B2-h3 within human framework sequences, including the V48L mutation);

- SEQ ID No: 11 (CDRs of N 13B2-h3 within human framework sequences, including the F87Y mutation);

- SEQ ID No: 12 (CDRs of N13B2-h3 within human framework sequences, including both the V48L and F87Y mutations); and

- an antigen-binding fragment thereof, particularly said fragment comprising the three CDRs consisting of the sequences set forth in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No: 6, more particularly a fragment comprising at least 74 amino acids consisting of amino acids 24 to 97 of SEQlDNo:12.

[10] Also provided herein are polynucleotides encoding the polypeptides provided herein, in particular the antibodies or antigen-binding fragments thereof of the invention. Such polynucleotides encode in particular polypeptides comprising or consisting of a sequence selected among the group consisting of SEQ ID Nos:9 to 12, and also encode polypeptides comprising or consisiting of sequences selected among SEQ ID Nos:l to 3, particularly SEQ ID No: 7, and may also encode polypeptides comprising or consisiting of sequences selected from the group consisting of SEQ ID Nos 26 to 28. In particular, said polynucleotides comprise or consist in the combination of at least two isolated nucleic acid sequences (molécules), a first isolated nucleic acid molécule comprising or consisting of a sequence selected among the group consisting of SEQ ID Nos: 15 to 18; in particular SEQ ID No. 18; said combination also comprising or consisting of a second isolated nucleic acid molécule comprising or consisting of a sequence selected from the group consisting of SEQ ID No: 13 and SEQ ID Nos: 29 to 31, in particular SEQ ID No. 13.

[11] In particular, the polypeptide according to the invention comprises an antibody light chain variable domain or an antibody light chain, said polypeptide comprising at least 84 % and more particularly at least 85 % of human residues. Said polypeptide can be an antigen-binding fragment and/or an antibody-mimetic molécule from an antibody disclosed herein.

[12] Provided herein are also compositions comprising said polypeptides, in particular antibody light chain variable domains or antibody light chains, antibodies, antigen-binding fragment or antibody mimetic molécule thereof, methods of obtaining said antibodies, nucleic acid molécules encoding said polypeptides and antibodies and uses of said polypeptides, antibodies and compositions.

[13] Accordingly the polypeptides, antibody light chain variable domains, antibody light chains, antibodies, antigen-binding fragments, antibody mimetic molécules and composition provided herein may be intended and/or suitable for use in order to remedy to a condition diagnosed in a human patient which results from pathogenesis related to lymphopoiesis, when IL-7 signalling pathways provide contribution to said pathogenesis, especially when an increase in the maturation, more precisely the upregulation of costimulatory molécules, of dendritic cells is undesirable.

Detailed description of the invention

Biochemistry

[14] CD 127 is common to the 1L-7 receptor (IL-7R) and to the TSLP receptor (TSLPR). The 1L-7R is constituted of a heterodimer of CD127 and the common gamma chain (yc) of interleukin receptors. The common gamma chain yc is sometime referred to herein and in the literature as CD132. IL-7R is bound by Interleukin 7. The TSLP receptor is a heterodimer of CD 127 and cytokine receptor-like factor 2 (CRLF2). The TSLP receptor is bound by TSLP. In the literature, TSLPR is sometimes used to designate both the CRLF2 chain of the receptor, and the CD127/CRLF2 complex. In order to avoid confusion, in what follows TSLPR usually désignâtes the complex.

[15] CD 127 (Swiss Prot accession number PI6871 ) may exist in four isoforms. The canonical isoform, also termed H20 (Swiss Prot P16871 .1 ) is a single-pass transmembrane protein and has 459 amino acids consisting, from N- to C-terminal, of a 20 amino-acid signal peptide, a 219 amino acid extracellular domain, a 25 amino-acid transmembrane domain and a 195 amino-acid intracellular domain. Other isoforms share the sequence of ail of (or most of) the extracellular domain of H20 and display varied C-terminal sequences. Isoforms 2 and 4 are secreted (Swiss Prot P16871 -4 and P16871 -3), while isoform 3 (Swiss Prot P16871-2) is also a transmembrane protein. CD127 is reported to hâve the sequence of SEQ ID No: 21, and its extracellular domain, when the signal peptide is removed, has the sequence of SEQ ID No: 22. Unless otherwise stated, the numbering used herein for amino acids of CD127 is the numbering from SEQ ID No: 22.

[16] CD127 is a Cytokine Receptor Homology class 1 (CRH 1) receptor. As is well known in the art, the extracellular domain of these receptors consists of two fibronectin 3 domains, termed DI and D2. The précisé crystallographic structure ofCD127 has been published and discussed in e.g. McElroy et al., 2009; McElroy et al., 2012 and Walsh, 2012 and in particular has been disclosed as protein structure data in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) database, with the accession number 3UP1. DI is generally considered to be involved in the binding with 1L-7, while D2 is involved in the binding to the yc chain (and also with 1L-7). Importantly, the site 2b of domain D2 comprises three beta sheets including the following sequences SEQ ID No. 32 (FDLSVIYRE); SEQ ID No: 33 (NDFWTFNTS) and SEQ ID No: 34 (TKLTLLQR). The site 2b of domain D2 is therefore included between amino acids 109 and 180 ofSEQ IDNo: 22(see Walsh, 2012; Verstraete, K. et al., Nature Com 2017). The site 2b of domain D2 may also be defined as being included between amino acids 109 and 173 of SEQ ID No. 22. The site 2b of domain D2 may also be defined as being included between amino acids 113 and 180 ofSEQ ID No. 22. The site 2b of domain 2b may also be defined as being inlcuded between amino acids 113 and 173 of SEQ ID No. 22. Particularly, the site 2b of domain D2 comprises, in particular consists essentially of, amino acids 109 to 133 of SEQ ID No: 22, in particular 109 to 127, wherein the first two beta sheets are localized; and amino acids 166 to 180 of SEQ ID No: 22, wherein the third beta sheet is localized. More particularly, the 2b site consists essentially of amino acids 113 to 133, in particular 113 to 127, of SEQ ID No: 22 and of amino acids 166 to 180 of SEQ ID No: 22. More particularly, the 2b site consists essentially of amino acids 109 to 133, in particular 109 to 127, of SEQ ID No: 22 and of amino acids 166 to 173 of SEQ ID No: 22. More particularly, the 2b site consists essentially of amino acids 113 to 133, in particular 113 to 127, of SEQ ID No: 22 and of amino acids 166 to 173 of SEQ ID No: 22. The site 2b of domain D2 is critical for the CD127-yc interaction, in particular to allow or increase binding of CD127 with yc in the presence of 1L7. In particular, mutations at PI 12 and L115, which hâve been identified in patients suffering from Severe combined immunodeficiency (SCID), are thought to destabilize the hydrophobie core of the D2 domain which likely results in their pathogenic feature. As said above, the 2b site consists essentially of amino acids 109 to 180 of SEQ ID No: 22, or consists essentially of amino acids 109 to 173 of SEQ ID No. 22, or consists essentially of amino acids 113 to 180 of SEQ ID No. 22, or consists essentially of amino acids 113 to 173 of SEQ ID No. 22. The skilled person will appreciate that the extremities of such a domain may not necessarily be defined unambiguously with a single-base précision and that the 2b site may be understood to comprise, at either or both ends of the mentioned sequence(s), 1,2, or 3 more or less amino acids. Therefore, when referring herein to the 2b site of CD127, this should be understood to refer to a sequence of CD127 starting at position 106, 107,108,109,110,111,112 or 113 and endingat position 173, 174, 175,176, 177, 178, 179 orl80 ofSEQ IDNo: 22; in particularto such a sequence which is thought or shown to constitute an essential binding site with the yc chain of the 1L7R, in particular in the presence of IL-7. More particularly, when referring herein to the 2b site of CD127, this should be understood to refer to a sequence of CD127 starting at position 106, 107, 108, 109, 110, 111, 112 or 113 and ending at position 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 of SEQ ID No: 22, but also to a sequence of CD 127 starting at position 162, 163, 164, 165 or 166 and ending at position 173, 174, 175, 176, 177, 178, 179 or 180 ofSEQ ID No: 22. It should be noted that the three beta sheets as defined herein may comprise epitope sequences spécifie for antibodies or antigen-binding fragments or antigen-biding mimetic according to the invention. Antibodies, antigen-binding fragments and antigen-binding mimetics according to the invention may be able to specifically bind to SEQ ID No: 32, SEQ ID No: 33 and/or SEQ ID No: 34. Furthermore, an amino acid sequence comprising at least a portion of one beta sheet and some contiguous amino acids localized at one end of any beta sheet may also be an epitope sequence spécifie for an antibody according to the invention. As an example, the sequence SEQ ID No: 35 (TLLQRKLQPAAMYEI) comprises the last five amino acids ofthe third beta sheet and ten extra amino acids localized outside the third beta sheet. As another example, the SEQ ID No. 96 (RKLQPAAM) comprises one amino acid localized within the third beta sheet and seven extra amino acids localized outside the third beta sheet. In particular, the antibody or antigen-binding fragment thereof or antigen-binding mimetic according to the invention may specifically bind to RI 73 of SEQ ID No: 22. In particular, the antibody or antigen-binding fragment thereof or antigen-binding mimetic according to the invention may specifically bind to at least one sequence selected from the group consisting of SEQ ID No: 32; SEQ ID No: 33; SEQ ID No: 34, SEQ ID No. 35 and SEQ ID No. 96. In particular, the antibody or antigen-binding fragment thereof or antigen-binding mimetic according to the invention may specifically bind to at least one sequence selected from the group consisting of SEQ ID No: 36 (LVEVKCLNFR); SEQ ID No: 37 (ICGALVEVKCLNFR) and SEQ ID No: 38 (LVEVKCLNFRK). Altematively or complementarily, the antibody or antigen-binding fragment thereof or antigen-binding mimetic according to the invention may specifically bind to at least one sequence selected from the group consisting of SEQ ID No: 39 (KKFLL1G); SEQ ID No: 40 (KKFLL1GKSN1) and SEQ IDNo: 41 (FIETKKFLL1G). SEQ IDNo: 36 to SEQ IDNo: 41 are localized within the domain DI of CD127 and are epitope sequences recognized by an antibody or antigen-binding fragment thereof or an antibody mimetic molécule according to the invention. In an alternative or complementary embodiment of the invention, the antibody or antigen-binding fragment thereof or an antibody mimetic molécule according to the invention may specifically bind to at least one sequence selected from the group consisting of SEQ ID No: 42 (CLNFR) and SEQ ID No: 43 (FIETKKF). These two sequences are epitope sequences localized within the domain DI of CD127. In a particular embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic according to the invention may specifically bind to at least one sequence selected from the group consisting of SEQ IDNo: 32; SEQ IDNo: 33; SEQ ID No: 34, SEQ IDNo: 35 and SEQ IDNo. 96, in particular from the group consisting of SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 96; and at least one sequence selected from the group consisting of SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40; SEQ ID No: 41; SEQ ID No: 42 and SEQ ID No: 43, in particular from the group consisting of SEQ ID No. 42 and SEQ ID No. 43. In a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically bind to SEQ ID No 34 and at least one sequence selected from the group consisting of SEQ ID No. 42 and SEQ ID No. 43, in particular to SEQ ID No. 42 and SEQ ID No. 43. In a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically bind to SEQ ID No: 35 and at least one sequence selected from the group consisting of SEQ ID No: 42 and SEQ ID No: 43, in particular to SEQ ID No. 42 and SEQ ID No. 43. In a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically bind to SEQ ID No: 96 and at least one sequence selected from the group consisting of SEQ ID No: 42 and SEQ ID No: 43, in particular to SEQ ID No. 42 and SEQ ID No. 43. In a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically bind to both sequences SEQ ID No: 35 and SEQ ID No: 96. In a preferred embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically bind to both sequences SEQ ID No: 42 and SEQ ID No: 43. In a particular embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic according to the invention may specifically recognize at least the third beta sheet of the site 2b of domain D2 of CD127. The third beta sheet is preferentially defined as being loçalized between amino acids 166 and 173 of SEQ ID No: 22, ^J corresponding to SEQ ID No. 34, and more particularly the third beta corresponds to SEQ ID No. 34.

In a more particular embodiment of the invention, the antibody or antigen-binding fragment or antigenbinding mimetic according to the invention may specifically recognize at least two of the beta sheets loçalized within the site 2b of domain D2, and more particularly, the antibody or antigen-binding fragment or antigen-binding mimetic according to the invention may specifically recognize the three beta sheets loçalized within the site 2b of domain D2 as defined hereabove. In a more particular 10 embodiment of the invention, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically recognize at least one beta sheet loçalized within site 2b of domain D2, in particular the third beta sheet corresponding to SEQ ID No. 34, and bind to at least one sequence selected from the group consisting of SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40; SEQ ID No: 41; SEQ ID No: 42 and SEQ ID No: 43. In a preferred embodiment of the invention, the 15 antibody or antigen-binding fragment or antigen-binding mimetic may specifically recognize at least the third beta sheet corresponding to SEQ ID No: 34, and at least one sequence selected from the group consisting of SEQ ID No: 42 and SEQ ID No: 43, in particular to SEQ ID No. 42 and SEQ ID No. 43. In a more particular embodiment of the invention, the antibody or antigen-binding fragment or antigenbinding mimetic may specifically recognize a linear peptide or a linear epitope consisting of amino acids 20 of SEQ ID No: 35. Altematively or complementarely, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically recognize a conformational peptide or a conformational epitope consisting of the amino acids of sequence ID No. 96. In a more particular embodiment, the antibody or antigen-binding fragment or antigen-binding mimetic may specifically recognize a linear epitope or a linear peptide of the amino acid sequence of SEQ ID No: 35, and a conformational epitope 25 or a conformational peptide of SEQ ID No: 96.

[17] 1L-7R signalling. Binding of 1L-7 to lL-7Rtriggers the activation of several signalling pathways, including the Janus kinases (JAK) -1 and -3, signal transducer and activator of transcription 5 (STAT5) and phosphatidylinostol 3-kinase (PI3-k). STAT1 and STAT3 pathways are reported to be activated, although they do not seem to be the main pathways. The activation of the STAT5 pathway is required 30 for the induction of the anti-apoptotic protein Bcl-2 and the prévention of the entry of the pro-apoptotic protein Bax in the mitochondrion and thus for survival of thymie developing T cell precursors. The activation of the PI3-k pathway results in the phosphorylation and cytoplasmic rétention of the proapoptotic protein B ad.

[18] TSLPR signalling. Thymie Stromal Lymphopoietin, (TSLP) is an épithélial Cell Cytokine that 35 is active in lymphopoiesis and in particular is involved in régulation of development of cells of the immune System, said régulation impacting in particular the maturation of said cells. Human TSLP (Genbank accession number AF338732) is a factor which exerts polarization of dendritic cells and promotes T and B cell prolifération and différentiation. TSLP also suppresses the génération of Treg cells (Lei et al., 2011 ).

[19] TSLP-induced signaling pathways hâve been shown to be different, at the molecular level, from IL-7-induced pathways. In particular, while TSLP binding to its receptor also activâtes Jak-1 , it does not activate Jak-3 but does activate Jak-2. These différences are consistent with the observation that Jak1 associâtes with CD127, shared by both receptors while Jak-2 associâtes with CRLF2 and Jak-3 with yc (Rochman et al., 2010). The activation of the STAT5 pathway is also reported for TSLP-induced signaling (Zhong et al., 2014). One major effect of TSLP is to lead to the activation of dendritic cells, inducing the overexpression of costimulatory molécules such as CD80, thereby promoting ΊΉ-2 mediated inflammatoiy responses (Reche et al., 2001 ).

Cellular biology

[20] CD127-posititive cells désignâtes cells expressing CD 127 at their cell surface. In most cases, CD127-positive cells express CD127 in a complex forming the IL-7R (lL-7R-positive cells) and / or in a complex forming the TSLPR (TSLPR-positive cells). CD127 is expressed by various cells, including by both memory and naïve T cells. CDI27 is in particular expressed by effector T cells (Teff), including resting and memory T cells, and by immature B cells, but is especially not expressed by resting natural regulatoiy T cells (natural Treg). CD 127 is essentiel for promoting thymocyte différenciation and clonal expansion of lymphocytes.

[21 ] The importance of the IL7-CD127 pathway for naïve T-cell homeostasis is underlined by several recent studies showingthat expression levels of membrane-bound CD127 on conventional CD4⁺ T cells correlate with frequencies of recent thymie émigrant (RTE)-CD4⁺ T cells in healthy individuals and HIV-infected patients as well as in patients with MS (Albuquerque et al., 2007) (Broux et al., 2010). CD127 is also a component of the TSLP receptor. The sécrétion of TSLP by Hassall's corpuscles, structures composed of épithélial cells in the thymie medulla, has been demonstrated to condition CD 11 c⁺myeloid dendritic cells (MDCs) to induce the différentiation of thymocytes into Treg ( Watanabe et al., 2005a). Accordingly, signais from the IL-7 receptor are required for Treg development as shown in CD 127 knockout mice (Mazzucchelli et al., 2008). In (Haas et al., 2011), the authors showed a reduced CD 127 expression on conventional T cells and upregulated IL-7 plasma levels together with réduction of recent thymie emigrant-Treg frequencies and Treg function in MS, without clear genetic influence (Haas et al., 2011).

[22] Dissecting how IL-7 régulâtes its cognate receptor membrane trafficking is crucial to the indepth understanding of the rôle of IL-7/IL-7R in lymphocyte function. Previous studies hâve suggested that IL-7 stimulation of T cells leads to surface down-modulation of CD127 within 30 minutes, possibly because of receptor intemalization. At later time points (2-6 hours), 1L-7 was shown to induce transcriptional down-regulation of CD127. However, the actual dynamics of CD127 intemalization and therégulation oftrafifickingmechanisms by 1L-7remaintobeelucidated(Henriquesétal.,2010). It was also suggested that lL-7-induced signaling is dépendent on CD 127 intemalization and that subséquent receptor dégradation relies on JAK3 activity and is mediated by both protéasomes and lysosomes.

Physiopathology

[23] Dendritic cells express high levels of costimulatoiy molécules after maturation, such as CD80, which promûtes T cell mediated immune responses. They also produce the cytokine TARC (CCL17), which induces chemotaxis in T cells. As such, mature dendritic cells contribute to the physiopathology of several immune-mediated diseases where T cell responses are at play, as for example in asthma, rheumatoid arthritis, colitis, multiple sclerosis and uveitis. Mature dendritic cells also play a key rôle in the rejection process of cells, tissues or organ allografts. Therefore, many therapeutic strategies aim at preventing dendritic cells maturation.

[24] The presence or absence of costimulatory molécules on antigen-presenting cells (APCs), such as dendritic cells signifïcantly influences the qualitative and quantitative nature of an immune response. Overexpression of CD80 by dendritic cells causes DC maturation and increases memory T cell activation (Bour-Jordan et al., 2011). Mechanistically, interaction of CD28 with CD80 occupies the central cluster of the immunological synapse and is colocalized with the engaged TCR, thereby stabilizing the immune synapse (Dustin and Shaw, 1999) (Grakoui et al., 1999). The interaction between CD28 and CD80 actually generates the appropriate spacing for TCR to effîciently interact with HLA molécules (Shaw and Dustin, 1997).

[25] Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). The appearance of demyelinating patches in the CNS of patients with MS is associated with an inflammatory infiltrate mainly composed of macrophages and T lymphocytes. On a mechanistic level, the MS is considered as an autoimmune disease. MS is typically considered as a disease primarily mediated by CD4 + T cells. Particular subsets of CD4 +: Thl and more recently Thl7, were implicated in the pathophysiology of the disease. At présent, it is still difficult to assign spécifie rôles to each subpopulation Thl and Th 17. Furthermore, inhibition of leucocyte trafïicking by antagonism of the alpha4 (a4)-integrin has now been validated as a therapeutic approach for the treatment of inflammatory diseases such as MS and inflammatory bowel disease (IBD) and as well for the treatment of atherosclerosis (Zhi et al., 2014). α4β7 is expressed on a more restricted set of leucocytes including activated macrophage, subsets of lymphocytes, NK cells, mast cells and eosinophils.

[26] Human IL-7 induces strong expression of a4 and β7 integrins in vitro on human T lymphocytes and dramatically increases the frequency of human T lymphocytes expressing α4, β7 and α4/β7 integrins, which are required for T lymphocytes homing and rétention in non-lymphoid tissues such as intestine, brain and skin (Denucci et al., 2009; Gorfu et al., 2009).

[27] Naïve T cells are partly responsible for acute rejection of transplanted organs and tissues. These cells can be controlled by current immunosuppressive drugs (calcineurin inhibitors) and by monoclonal antibodies that block costimulation (anti-adhesion, CD80/86 inhibitors). Memory T cells are also responsible for transplant rejection. Memory T cells accumulate in man due to the acquired immune history, mainly former reactions against viruses. It has been shown that memory T cells can be reactivated by alloantigens as a resuit of “heterologous immunity”, which is the cross-reaction of our anti-viral defenses with alloantigens (Adams et al., 2003). Heterologous immunity represents a potent barrier to tolérance induction since memory T cells, in contrast to naïve T cells, are programmed to activate quickly, with a reduced requirement for costimulatory signais. Memory T cells may also be involved in chronic rejection. Beside their rôle in organ and tissue transplantation, naïve and memory T cells are also co-responsible for many autoimmune diseases. This is the case for ulcerative colitis (Shinohara et al., 2011), rheumatoid arthritis, psoriasis or graft-versus-host disease.

[28] Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD), are chronic relapsing gastrointestinal disorders characterized by chronic intestinal inflammation, dysregulated immune responses to intestinal microbiota and dysfunction of the épithélial barrier (Khor et at., 2011; Abraham and Cho, 2009). The incidence and prevalence rates of IBD are increasing worldwide and these diseases are associated with marked morbidity and hâve a major impact on quality of life and ability to work (Danese and Fiocchi, 2011; Baumgart and Sandbom, 2012). Current conventional treatments aim at dampening inflammation with the graduai use of anti-inflammatory agents, immunosuppressive drugs and biological agents targeting înflammatoiy cytokines such as tumor necrosis factor alpha (TNFa). A key feature of IBD is also the rapid recruitment and prolonged persistence of leukocytes at the site of inflammation, which is permitted by integrins interaction Tvith cognate receptors expressed by endothélial cells allowing cell adhesion and transmigration (Adams and Eksteen, 2006; Agace, 2006). Emerging thérapies are targeting this entry-door to the gut with antiadhesion molécules, specially targeting the gut-specific α4β7 integrins pathway (Feagan et al., 2013; Sandbom et al., 2013)⁷·⁸. However, these thérapies do not maintain remission in more than half of patients, relapsing flares occurring in a high proportion of primary responders. Opportunistic infections also develop as conséquence of general immunosuppression. Thus, one major goal is to provide novel treatments for IBD and to identily markers which détermines the chronicity, response and relapse of IBD.

[29] Furthermore, several malignant cells hâve been shown to display IL-7R. This is the case for Sezary cutaneous lymphoma (60% of them), or childhood acute lymphoblastic leukemia in which about 15% of the cases develop gain-of-function mutation in CD 127, rendering these tumors partially IL-7 dépendent (Shochat et al., 2011).

[30] The déplétion of T lymphocytes has been an obvious immunosuppressive approach to counteract allograft rejection or fight autoimmunity. However, total T cell déplétion might not be favorable for the induction of immunological tolérance. Targeting T cell subpopulations or selectively activated T cells, without modifying Treg cells, could constitute a pro-tolerogenic approach (Haudebourg et al., 2009). CD 127 may thus be regarded as a potential attractive therapeutic target for monoclonal antibodies (Mabs) aimed at modulating immune responses since such monoclonal antibodies could hâve the potential of depleting effector but not regulatory lymphocytes. It has been assumed accordingly that they might show efficacy in transplantation, autoimmunity (Michel et al., 2008) and malignancies by antagonizing access of 1L-7 to IL-7-R and thereby limiting T and B cell fonction and growth.

[31] A therapy with a monoclonal antibody against CD127⁺ cells that interfères with the 1L-7 pathway could fulfili that goal by eiiminating/neutraiizing naïve and memory T cells and/or reducing their number while preserving Treg cells or by eliminating or reducing the number of CD127-positive malignant cells. A therapy with a monoclonal antibody against CD127⁺ cells might however act as a double edge sword if it leads to dendritic cells activation. Indeed, CD 127 is also expressed by dendritic cells in association with CRLF2, forming the TSLP receptor. in the presence of TSLP, dendritic cells get activated and promote T cell-mediated immune responses. Some monoclonal antibodies against CD127, presumably by modifying the way TSLP interacts with TSLP receptor, hâve the property to increase the maturation of dendritic cells înduced by TSLP. As a conséquence, a therapy with a monoclonal antibody against CD127 that would not increase the maturation of dendritic cells induced by TSLP would présent a therapeutic advantage. It would présent the benefit of IL7R blockade without the drawback of activating dendritic cells in an inflamed environment containing TSLP.

[32] In a publication (Racapé et al., 2009) the authors analysed the interest ofIL-7 receptor alpha (IL7Ra) as a potential therapeutic target in transplantation. Having reviewed the expression of IL-7Ra on varions T cells and IL-7 responsive cells, the authors determined whether targeting memory T cells expressing IL-7Ra could prolong allograft survival in mice and conclude that targeting IL-7 or IL-7Ra would advantageously spare Treg cells. Among the perspectives, the authors pointed out that targeting either IL-7 or IL-7Ra in therapeutic treatment might hâve different conséquences on the survival of the cells expressing CD 127 and might elicit different types of lymphopenia. The question of the effects of antibodies that would be directed against IL-7Ra depending upon whether they would be blocking or neutralizing or cytotoxic antibodies was also posed from a conceptual point of view. The authors nevertheless did not show having obtained and assayed such antibodies and rather expressed the need for further study to assess the relevancy of the hypothesis.

[33] In view of the drawbacks of available therapeutic approaches in immune reiated diseases and other diseases involving the IL-7/IL-7Ra such as different types of cancers, including some breast cancers, there is still a need for further drug candidates, especially for candidates active with respect to more sélective targets for the purpose of controlling e.g. modulating immune activation in human patients.

[34] In this context, monoclonal antibodies against IL-7Ra having antagonist properties toward IL7Ra hâve been disclosed in WO2010/017468 and their humanized versions in WO2011/094259 with a view to treat autoimmune diseases like multiple sclerosis. The described antibodies are said to be antagonist for IL-7 binding to its receptor, and active against Th17 and ThI cells expansion and survival which were said to require IL-7 interaction with their CD 127 receptor. The effect of these antibodies on the maturation of immune cells, and particularly of dendritic cells, has not been considered. Besides, these antibodies are said not to inhibit TSLP-induced production of TARC (p. 107 of WO2011/094259). Similarly, anti-CD127 antibodies reported in WO2011/104687 or in WO2013/056984, which are contemplated for use in the treatment of diabètes, lupus, rheumatoid arthritis and other autoimmune diseases, hâve not been discussed with respect to their possible effect on the maturation of dendritic cells and their interaction with TSLP-induced signaling has not been reported. In addition, as published by Kern et al (Kern et al., 2013; Kern et al., 2015) and as shown herein, the anti-CD127 antibodies of the prior art induce intemalization of the receptor. Since antagonist anti-CD127 antibodies that also induce intemalization of CD127 fait to control cutaneous type IV hypersensitivity, whereas antagonist antiCD 127 antibodies that do not induce intemalization do, it might be that the intemalization process activâtes the signaling pathway, mitigating the antagonist effect of the antibodies. Last, the antibodies of the prior art recognize an epitope which does not comprise any sequence from the 2b site of CD127 (i.e. in particular from amino acids 109-180 of SEQ ID No:22, or in particular from amino acids 109173 of SEQ ID No. 22, or in particular from amino acids 113-180 of SEQ ID No. 22, or in particular from amino acids 113-173 of SEQ ID No. 22); and hâve not been shown to disrupt the binding of CD127 with the yc chain of the IL7-R.

[35] Despite recent interest in the development of CD127 antibodies, efforts hâve thus concentrated on the inhibition of lL7-induced 1L-7R signalling. Nonetheless, TSLP and the TSLPR hâve been involved in a number of pathologies. TSLP has been shown to play a rôle in skin and lung diseases (He and Geha, 2010) and to associate to various pathologies including airway inflammatory disease and atopie dermatitis in human and mice (Ying et al., 2008) (Jariwala et al., 2011). In addition TSLP has been shown to associate to régulation of intestinal immunity and inflammation (Taylor et al., 2009). Other pathologies involving TSLP and the TSLPR include pédiatrie B-cell leukemia (van Bodegom et al., 2012), lung- and skin- spécifie allergie disorders, autoimmunity-related diseases (Roan et al., 2012) and cancer, including breast cancer (Olkhanud et al., 2011).

[36] Antibodies that do not display the effect of increasing the maturation of dendritic cells and/or that do not induce intemalization of CD127 and/or that inhibit IL7-induced intemalization were disclosed in WO 2015/189302. Said antibodies,termedN13B2(chimericantibody),N13B2-hl,N13B2h2 and N13B2-h3 (humanized N13B2) in said application and hereinafter, hâve high efficiency, especially in vivo and in particular were shown to hâve a fast effect on effector memory T cells, as defined hereinafter. However, said antibodies are humanized to a limited extent and their production efïïciency is also limited. Moreover, no long-lasting effect of said antibodies on effector memory T cells has been reported.

[37] The invention relates to tools for the design of novel antibodies suitable as therapeutic candidates for the administration to patients with or at risk of a disease involving 1L-7 signaling pathways. According to various approaches provided herein, such tools improve préparation of antibodies intended for administration to human hosts. Such tools comprise in particular humanized, particularly monoclonal, antibodies which comprise ail the CDRs sequences ofN13B2-h3.

[38] Such polypeptides (or polynucleotides encoding such polypeptides) are provided in particular for the production of antibodies (or antigen-binding fragments thereof) and/or as antibodies (or antigenbinding fragments thereof), particularly which specifically binds to CD127; said antibodies, in particular monoclonal antibodies, comprise a heavy chain variable domain comprising the three CDRs sequences VHCDR1 set forth in SEQ ID No: 1, VHCDR2 set forth in SEQ ID No: 2 and VHCDR3 set forth in SEQ ID No: 3. In particular, said heavy chain variable domain consists of the sequence set forth in SEQ ID No: 7 or has said sequence with additional mutations, in particular substitution, délétion or insertion of four residues, preferably three or two residues and even more preferably one residue, preferably wherein said mutations are neither in the CDR sequences nor in canonical or Vernier positions of the framework sequences.

[39] The antibodies provided herein are preferably monoclonal antibodies, meaning that a composition of these antibodies is homogeneous, especially identical, in tenus of antigen-binding specificity and accordingly in terms of variable région composition.

[40] The invention provides antibodies which share the CDRs of the N 13B2-h3 antibody but hâve a distinct light chain variable domain framework. The inventors hâve surprisingly shown that antibodies comprising the same CDRs of the light chain variable domain of N13B2-h3, i.e. the CDRs with SEQ ID No: 4 and 5 and 6 but comprising a different light chain variable domain sequence, i.e. of SEQ ID No: 9; of SEQ ID No: 10; of SEQ ID No: 11 or of SEQ ID No: 12; while humanized extensively, are highly produced compared to N 13B2-h3 (up to 4 times higher), while preserving ail the functional features.

[41] The invention provides means suitable in this context, comprising in particular spécifie monoclonal antibodies against IL-7Ra. In particular embodiments, said antibodies interfère only negatively with the TSLP pathway. Accordingly, in preferred embodiments said antibodies do not increase TSLP-induced dendriticcell maturation. In addition oraltematively, in particular embodiments, said antibodies do not induce intemalization of CD 127. and/or inhibit lL7-induced internalization of CD 127. In particular embodiments, said antibodies combine these DC maturation- and/or intemalization-related properties with antagonist activity toward IL-7/IL-7-R signalling. In particular embodiments, said antibodies inhibit lL7-induced expression of α4, β7 and α4/β7 integrins in T cells, in particular in vivo. In particular embodiments, said antibodies exert a cytotoxic action against target

CD127+ cells that physically reduce their number (contraction of the subpopulation). In addition or alternatively, in particular embodiments said antibodies comprise at least 80 %, preferably at least 84 %, or more than 84 % and even more preferably at least 85 % of human residues as defined hereinafter. In addition or alternatively, in particular embodiments said antibodies are produced at least as efficiently as N13B2-h3, preferably at least twice as efficiently, as defined hereinafter. In addition or alternatively, in particular embodiments said antibodies hâve rapid and/or a long-lasting effect on effector memory T cells.

[42] In particular, the antibodies provided herein comprise a variable heavy (VH) chain comprising the following amino acid sequences:

V HCDR1 SEQ lDNo:l;

V HCDR2 SEQ IDNo: 2;

V HCDR3 SEQ ID No: 3.

The antibodies provided herein preferably comprise a VH chain consisting of the sequence set forth in SEQ ID No: 7:

QVQLVESGGGLVKPGGSLRLSCAVSGFTLSDYYMAWIRQAPGKGLEWVST1SASGLRTYYPD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLSAHYGFNYFDYWGQGTLVTVSS. Said variable heavy chain is in particular linked to the constant heavy chain consisting ofthe sequence of SEQ ID No: 26 to constitute a complété antibody heavy chain.

[43] In particular, the antibodies provided herein comprise a VL chain consisting of a sequence selected from the group consisting of the sequence of SEQ ID No: 9, SEQ ID No: 10, the sequence of SEQ IDNo: 11, and the sequence ofSEQ IDNo: 12, in particular the sequence ofSEQ IDNo: 12:

DIQMTQSPSSLSASVGDRVT1TCRTSED1YQGLAWYQQKPGKAPKLLLYSANTLH1GVPSRFSG SGSGTDYTLTISSLQPEDFATYYCQQYYDYPLAFGGGTKVE1K. Said variable light chain is in particular linked to the constant light chain consisting of a sequence selected from SEQ ID No: 27 and SEQ ID No: 28, in particular SEQ ID No: 27, to constitute a complété antibody light chain.

In particular, the antibody or antigen-binding fragment thereof according to the invention, specifically binds to CD127, in particular to human CD127 and comprises:

In a more particular embodiment of the invention, the antibody or antigen-binding fragment thereof, specifically binds to CD 127, in particular to human CD 127 and comprises:

- an antibody light chain variable domain or an antibody light chain according to the invention, particularly consisting of a sequence selected from the group consisting of SEQ ID No: 9, SEQ ID No: 10; SEQ ID No: 11 and SEQ ID No: 12, in particular SEQ ID No: 12; and

Binding of CD127

[44] In accordance to the invention, “binding” to the IL-7Ra protein refers to an antigen-antibody type interaction and encompasses “spécifie binding” properties of the antibodies or antigen-binding fragments thereof which spécifie binding means that the antibodies or antigen-binding fragments bind to the IL-7Ra protein while they do not bind or bind with a significantly weaker affinity to other proteins (e.g. the common cytokine receptor γ-chain). Spécifie binding is preferably defined and/or determined in physiological conditions, especially in terms of pH and sait content of the testing solution. Binding and binding specifïcity can be assayed in accordance with the tests disclosed in the Examples and in particular can be assayed by biosensor, Blitz, Biacore, ELISA, or Western Blot analysis.

[45] In particular embodiments, the antibodies provided herein target and bind theIL-7-R alpha chain when it is complexed in the TSLP-Receptor (with CCRF2; Genbank accession Number AF338733; Reche et al., 2001). In particular embodiments, the antibodies provided herein bind to CD 127 as an isolated protein with an affinity constant (KD) equal to or lower than 5E-9 M, as may be determined by biosensor analysis, in particular by Blitz method. In particular embodiments, the binding properties of the antibodies are determined or defined using an antigen of human CD 127 comprising the sequences of epl (SEQ ID No: 19) and/or ep2 (SEQ ID No: 20). In particular embodiments, the antigen comprises a fragment of human CD127 comprising both epl and ep2 (i.e. the antigen comprises the sequences of epl and ep2 and the intercalated sequences of human CD127). In particular embodiments, the antibodies provided herein hâve at least the same affinity to said antigen as the N13B2-h3 antibody disclosed in WO 2015/189302, and/or as the N13B2-hVL6 antibody disclosed herein (with the VH having the sequence of SEQ ID No: 7 and the VL having the sequence of SEQ ID No: 12) and/or as the antibody with the VH having the sequence of SEQ ID No: 7 and the VL with the sequence of SEQ ID No: 10 or with the sequence of SEQ ID No: 11.

[46] Methods to test for the binding of the antibodies to their target, either full-length CD 127, isolated or as the TSLPR or IL7R, or an antigen thereof as above, comprising in particular the Blitz method, are known to the skilled person and are illustrated in particular in Figure 3, Example 3 and Example 4 herein and in p. 14, Figures 3,4 and 6 and the respective legends, and Examples 1,2,6,7 of WO 2015/189302.

Absence of increased TSLP-induced dendritic cell maturation

[47] The antibodies provided herein may bind CD127 in the TSLP receptor (i.e. may bind CD127 when it is in a complex with the CRLF2, forming the TSLP receptor). Therefore, the antibodies provided herein may interfère with TSLP-induced and / or TSLP receptor-mediated signaling. Preferably, the antibodies provided herein do not synergize with TSLP for the maturation of immune cells, in particular dendritic cells. In otherwords, the antibodies of the invention do not increase the maturation of immune cells induced by TSLP.

[48] This effect is particularly desired on the maturation of dendritic cells. The means to measure such effect are known to the skilled person and are disclosed in particular in WO 2015/189302 at pages 16-17 and in Example 9 thereof In particular, the antibodies provided herein do not increase expression of CD40 by more than 50% when compared to stimulation with TSLP alone (without antibody). Preferably, the expression of CD40 is not increased by more than 25 %, preferably not by more than 10 % and even more preferably not by more than 5%. In particularly preferred embodiments, the expression of CD40 is not increased or is decreased in cells stimulated with TSLP and with said antibodies when compared to cells stimulated with TSLP alone.

Inhibition of IL7-induced expression of α4, β7 and α4/β7 integrins

[49] In particular embodiments, the antibodies provided herein inhibit IL7-induced expression of a4, β7 and α4/β7 integrins in vitro. IL7-induced expression of α4, β7 and α4/β7 integrins, as used herein, désignâtes either or both the increase in the level of expression of a4 and β7 integrins and the increase in the number or ratio of T lymphocytes expressing α4, β7 and/or α4/β7 integrins. The inhibition may be partial, i.e. the level of expression of α4, β7 and α4/β7 integrins in the presence of IL7 is increased over baseline level (i.e. the level with neither antibody nor IL7) in the presence of antibodies, but less than in the absence of antibodies; or the inhibition may be complété, i.e. the level of expression of a4, β7 and α4/β7 integrins in the presence of 1L7 and of the antibody is no higher than baseline level.

[50] In particular embodiments, the antibodies provided herein inhibit expression of α4, β7 and/or α4/β7 integrins in vitro, i.e. the level of expression of α4, β7 and/or α4/β7 integrins is lower in cells treated with antibodies (and with and/or without 1L7) than in untreated cells (i.e. without antibody or 1L7). The extent of inhibition may be dose-dependent. The inhibition of expression may be more specifically defined, tested and/or measured as set forth in WO 2015/189302 in p. 18, in particular paragraph [58], and in the Example 16.

Inhibitors of CD127 intemalization

[51] In a particular embodiment, the antibodies provided heren inhibit the IL7-induced intemalization of CD 127. Thus, when incubated with said antibodies, the presence of IL7 induces no decrease in the cell surface expression of CD 127, or induces a less strong decrease in the cell surface expression of CD 127 than cells incubated without antibodies. In particular embodiments, when incubated with said antibodies, the level of CD 127 cell surface expression when cells are incubated at 37 °C for 15 minutes with 5 ng/mL 1L7 is at least 80 %, preferably at least 90 % of the cell surface expression level in cells incubated without IL7. In vitro, the cell surface expression is preferably measured after a limited time as indicated above. Besides, as most cellular intemalization processes are inhibited at low température, the effect is usually best observed at physiological température, in particular 37 °C. However, it is also contemplated to incubate cells at low température, in particular 4 °C.

[52] In a preferred embodiment, the antibodies provided herein do not induce the intemalization of CD127. Titus, the cell surface expression of CD127 in cells incubated in the presence of said antibodies is not reduced, or is not signifîcantly reduced, relative to cell surface expression in cells incubated in otherwise identical conditions, but in the absence of the antibody. In particular embodiments, when incubated at 37 °C for 30 to 45 minutes in the presence of 50 ng/mL of antibody, the level of CD 127 cell surface expression is at least 80 %, preferably at least 90 % of its level in cells incubated in the absence of the antibody. This effect may be observed in the absence of IL7 (in both antibody-treated and —untreated cells), in the presence of IL7, and/or both.

[53] The two CD 127 intemalization-related feature described above (i.e. inhibition of IL7-induced intemalization or non-induction of intemalization) may be further defined and/or tested as set forth in WO2015/189302 in particluar in paragraphe [59]-[63] at pages 19-20 and in Figure 16 and Example 5.

Disruption of CD127 -yc chain interaction

[54] According to a particular embodiment, the antibodies provided herein may disrupt the binding of CD 127 to the yc chain of the IL7-R. This means that, under conditions (in particular Chemical and physical conditions) where CD127 and yc chain are bound together in the absence of antibody, and in particular in the presence of IL7, the presence of said antibodies signifîcantly reduces said bond. In particular embodiments, in the presence of antibody and of IL7, CD 127 does not bind to yc. In particular, in the presence of the antibody and of 1L7, the amount of yc chain found associated with (or bound to) CD 127 is less than 80 %, preferably less than 50 %, even more preferably less than 25 % or 10 % of the amount bound in the absence of the antibody (or in the presence of another anti CD-127 antibody such as MD707-13) in otherwise identical conditions, in particular in the presence of IL7. Such a feature of the antibody may be assessed in particular by co-immunoprecipitation methods, well known to the skilled person for testing the interaction of proteins and illustrated e.g. in WO2015/189302 in Example 21. In particular, cells may be incubated in the presence or absence of the tested antibody, then solubilized in conditions allowing for the préservation of protein complexes, and the resulting lysate may bc subjccted to an anti-CD 127 immunoprécipitation and the presence ofyc in the CD127-containing immunoprecipitated complex assessed by western blotting using anti-yc antibodies (conversely, the immunoprécipitation may be performed using anti-yc antibodies and the presence of CD 127 assessed using anti-CD 127 antibodies).

Antagonist towards IL7-IL7-R interaction

[55] According to a particular embodiment, a macromolecule, in particular an antibody or antigenbinding fragment thereof, of the invention further has antagonist properties toward interleukin 7 (IL7) thereby antagonizing access, i.e. binding of 1L7 to CD127 on CD127 positive cells.

[56] “Antagonist properties towards IL7-IL7-R interaction” means that antibodies or antigen-binding fragments thereof of the invention, which target the IL7-Ralpha, hâve the effect of preventing the accessibility of the IL7 receptor expressed on CD 127 cells, especially human effector T cells, in particular human memory T cells, for its binding partner IL7, especially human IL7. As a resuit of antagonizing binding of 1L7, the antibodies of the invention or their functional fragments lead to lymphopenia by preventing IL7-dependent thymie T cells génération.

[57] Tire antagonist properties may be in particular antagonism toward IL7-R signaling induced by IL7. An antagonist of IL7-R signaling induced by IL7 can be identified by measuring the inhibition of STAT5 phosphorylation as described in the Examples. The IL7-induced phosphorylation of STAT5 is amarker of IL7-R activation and an antibody antagonizing IL7-IL7-R interaction is expected to decrease IL7-induced phosphorylation of STAT5.

[58] In particular embodiments, the macromolecule of the invention is an antagonist of IL7-R signaling induced by IL7. In a particular embodiment, the macromolecule of the invention inhibits 1L7induced phosphorylation of STAT5. In preferred embodiments, the inhibition of STAT5 phosphorylation is greater than 50 % at antibody concentrations as low as 55 ng/ml and / or the inhibition of STAT5 phosphorylation is greater than 80 % at antibody concentrations as low as 100 ng/ml. Inhibition of STAT5 phosphorylation may be assessed by methods known to the skilled person and in particular by the method set forth in the examples section, in particular in Example 5, and/or in page 21, paragraphs [69] and [70] and Example 3 of WO2015/189302.

[59] 1s is also désirable that the macromolecule of the invention (in particular antibody, antigenbinding fragment thereof or antibody mimetic molécule) inhibits the activation and/or does not activate or increase the activation, of the PI3-k and/or ERK (Extracellular signal-regulated kinase) signalling pathways and in particular inhibits the phosphorylation and/or does not induce or increase the phosphorylation of PI3-k and/or ERK 1 and/or ERK 2. In particular, the antibody, antigen-binding fragment thereof or antibody mimetic molécule provided herein, and in particular such antibody, antigen-binding fragment thereof or antibody mimetic molécule that is an antagonist towards IL-7 — IL7R interaction, does not induce the activation of the PI3-k and/or the ERK pathways (preferably of the PI3-k and the ERK pathway), and in particular does not induce the phosphorylation of PI3-k and/or of ERK 1 and/or ERK 2, more particularly does not induce the phosphorylation of PI3-k and of ERK 1 and of ERK 2. In particular, the antibody, antigen-binding fragment thereof or antibody mimetic molécule provided herein, and in particular such an antibody, antigen-binding fragment thereof or antibody mimetic molécule that is an antagonist towards IL-7 — IL-7R interaction, inhibits tire activation of the P13-k and/or the ERK pathways, and in particular inhibits the phosphorylation of P13-k and/or of ERK 1 and/or ERK 2, more particularly inhibits the phosphorylation of PI3-k and of ERK 1 and of ERK 2. The activation of the pathways and/or phosphorylation of said proteins, may be tested by methods known to the skilled person and in particular by Western blotting as illustrated in Figure 7 and Example 8.

Antagonist for binding of TSLP

[60] Since the antibodies provided herein bind CD 127 in the IL7-R, they may also bind CD 127 in the TSLPR and, particularly by steric hindrance and / or by compétition on common binding sites, they may inhibit the binding of TSLP to the TSLPR. In other words, the antibodies provided herein may présent antagonist activity for the binding of TSLP.

Inhibitor of TSLP-induced TARC production

[61] In a particular embodiment, the antibodies provided herein inhibit TSLP-induced TARC production of CDI27-positive cells. As mentioned above, TSLP-stimulated dendritic cells produce elevated levels of TARC. This may resuit from their binding to the TSLPR and their potential action as antagonists of TSLP binding. In a particular embodiment, the antibodies provided herein do not increase the maturation of dendritic cells (maturation being e.g. determined an increased expression of CD40 and/or CD80 cell surface marker).

[62] The level of TSLP-induced TARC production may be lower in cells treated with TSLP together with the anti-CD 127 antibodies prvoided herein than in cells treated with TSLP alone. In other words, the antibodies prvoided herein may be inhibitors of TSLP-induced TARC production. In an embodiment of the invention, the antibodies prvoided herein decrease the levels of TARC production. In a particular embodiment, the level of TARC production in cells treated with TSLP and an antibody provided herein is reduced by more than 10 %, preferably more than 20 %, compared to the level in cells treated with TSLP alone, at antibody concentrations as low as 1 pg/ml. Measurement of TARC production can be carried out on CD127-positive immune cells, in particular dendritric cells, from a blood sample using any standard method known from the skilled person and is illustrated e.g. in WO2015/189302 in Example 9.

Cytotoxic activity

[63] In a particular embodiment, the antibodies provided herein are cytotoxic against human cells, especially human T cells expressing CD127. Human cells expressing CD127 as a chain of 1L7 receptor, which are the target of said antibodies, are mainly T lymphocytes and more precisely are subpopulations of effector T lymphocytes including naïve and memory T cells but are not regulatory T cells (Treg), especially not resting natural Treg. Memory T cells are generated as a resuit of antigen priming and mainly defined by their functional characteristics, including ability to undergo recall prolifération upon re-activation and différentiation into secondary effector and memory cells. Similarly, the targeted TSLP receptor (as a complex including the IL-7-R alpha chain) régulâtes T helper lymphocyte, B cell and dendritic cell différentiation.

[64] In particular, the antibodies provided herein, having “cytotoxic activity against T cells” or cytotoxic properties (cytotoxic antibodies) give rise to déplétion in the effector T cell population by killing these cells and accordingly reduce the number of these cells when administered. Conversely, said antibodies do not alter the subpopulation of regulatory T cells or do not alter it to a significant extent, allowing the Treg cells to perform their function. In this context, in a particular embodiment, the ratio of regulatory T (Treg) versus effector T (Teff) cells raises following administration of said antibodies. In particular, the antibodies provided herein enable to raise said ratio of about 10% or more. In particular, the increase in the ratio of Treg versus Teff is of about 20%.

[65] In particular, the cytotoxic antibodies show Antibody-Dependant Cellular Cytotoxicity (ADCC). Alternatively, the antibodies provided herein hâve no ADCC properties. Antibody ADCC potential may be considered positive when spécifie cytoxicity is e.g. superior to 10%. ADCC properties can bc evaluated in an ADCC assay such as the test described in Example 10 of WO2015/189302. When the antibody is a rat antibody the effector cells used in the ADCC assay are preferably LAK (Lymphokine-activated killer) cells of rat. When the antibodies are humanized the ADCC assay can be carried out in particular on human NK cells.

[66] The antibodies of the invention which hâve both cytotoxic and antagonist properties for CD 127 positive cells enable cumulative effects of these properties with respect to the déplétion of effector T cells, especially of memory T cells, thereby enabling a stronger déplétion (exhaustion of the pool of CD 127+ cells) and corresponding réduction in the number of target T cells.

[67] The paragraphe above as well as the Examples describe how to test for the relevant desired functional characteristics of the antibodies provided herein. The following sections will detail varions structural characteristics and possible modifications of the antibodies. In light of these guidances, the skilled person will be able to obtain antibodies having the structural characteristics below along with the desired functional characteristics, in particular starting from the N13B2-hVL6 antibody which has the desired functional characteristics.

[68] When intended for administration to human subjects, it is désirable that antibodies présent as strong as possible a homology with human antibodies, as the skilled person is aware. The degree of identity with a human antibody is measured as the % of human residues in the antibody sequence, in particular in the framework of the antibody light or heavy chain variable domain i.e. the % of residues in tire antibody sequence, in particular in the framework of the antibody light or heavy chain variable domain, which are identical at the same fonctionnai position to the residue in the most homologous known human antibody, in particular in the framework of tire most homologous known human antibody light or heavy chain variable domain. This feature is generaly expressed herein, as in the littérature, as “antibody A (or the framework sequence of its variable domain) has xx % human residues”, which means that antibody A (or the framework sequence of its variable domain) has xx % of residues which are identical at the same fonctionnai position to the residues of the most homologous known human antibody (or the framework sequence of its variable domain). Such degree of identity may be measured by means known to the skilled person and in particular with the International Immunogenetics Information System (1MGT) DomainGapAIign tool as clarified during open session of WHO INN Expert Group (April 2015). Preferably, the antibodies provided herein hâve more than 80 %, even more preferably at least 84 %, more than 84 % and even more preferably at least 85 % identity with a human antibody, in particular as determined using the DomainGapAIign tool. Said degree of identity may be determined for the light chain variable domain only or for light chain only (by comparison with a human antibody light chain variable domain or light chain) or for the light chain and the heavy chain taken together, by comparison of each chain with the most homologous corresponding human antibody chain, and reporting the cumulated percentage of identity. Particularly, the antibody light chain variable domain or the antibody light chain according to the invention comprises at least 80%, more particularly 84 % and even more particularly at least 85 % of human residues. The particular antibody light chain variable domains provided herein hâve respectively 84.2 % (SEQ ID No: 9); 85.3 % (SEQ ID No: 10 and SEQ IDNo:l 1) and 86.3 % (SEQ ID No: 12) human residues. The preferred antibody heavy chain variable domain provided herein (SEQ ID No: 7) has 90.8 % human residues.

[69] An antibody light chain variable domain provided herein may comprise up to 135 amino acids, preferably up to 120 amino acids and even more preferably up to 110 or 107 amino acids. An antibody light chain variable domain provided herein may comprise at least 80, preferably at least 90 and even more preferably at least 100 or 106 amino acids. An antibody light chain variable domain provided herein may comprise 80 to 135, preferably 90 to 120 and even more preferably 105 to 110 amino acids.

[70] An antibody light chain provided herein may comprise up to 250 amino acids, preferably up to 230 amino acids and even more preferably up to 214 or 211 amino acids. An antibody light chain provided herein may comprise at least 150, preferably at least 190 and even more preferably at least 200 or 210 amino acids. An antibody light chain provided herein may comprise 150 to 250, preferably 190 to 230 and even more preferably 210 to 220 amino acids.

[71] Provided herein are also polypeptides which are (i) antigen-binding fragments of the antibodies provided herein, in particular antigen-binding fragments consisting or comprising a fragment of the antibody light chains or antibody light chain variable régions provided herein, an antibody heavy chain variable domain provided herein and/or (ii) antibody mimetic molécules of the antibodies provided herein.

[72] An antigen-binding fragment is a polypeptide which binds specifically to the CD 127 protein as defined above in the relevant section (paragraphs [44] to [47]) and comprises at least the three CDRs of N13B2-h3 (consisting of the sequences of SEQ ID Nos:4 to 6), preferably a fragment of the light chain variable domains provided herein (with SEQ ID Nos: 9 to 12) comprising said CDR sequences. An antigen-binding fragment may comprise at least 40, preferably at least 50 and even more preferably at least 70 or 74 amino acids. An antibody-binding fragment may comprise at most 100, preferably at most 90 and even more preferably at most 80 or 74 amino acids. An antigen-binding fragment may comprise 40 to 100, 50 to 90 and preferably 60 to 100 and even more preferably 50 to 70 or 60 to 80 amino acids. An antigen-binding fragment as provided herein may in particular hâve any of the suitable functional features disclosed in respect to the antibodies provided herein, in particular the features disclosed in paragraphs [47] to [62].

[73] Accordingly, an isolated polypeptide provided herein may consist of a sequence of up to 250 amino acids, in particular of up to 217, of up to 214, of up to 211, more particularly of up to 200, of up to 175, of up to 150, of up to 13 5, of up to 120, of up to 107 and even more particularly of up to 100, of up to 90, of up to 80, of up to 74, of up to 70, of up to 60 amino acids,

[74] An antibody mimetic molécule is a polypeptide with properties similar to an antibody, in particular with similar binding properties with CD 127. Antibody mimetics may be, for examples, affïbody molécules, affilins, affïmers, anticalins, monobodies, etc. An antibody mimetic molécule as provided herein may in particular hâve any of the suitable functional features disclosed in respect to the antibodies provided herein, in particular the features disclosed in paragraphs [47] to [62].

[75] In particular, the antibody according to the invention is a humanized antibody, which comprises constant domains derived from human constant domains. In particular, the antibody light chain constant domain is derived from a human kappa light chain constant domain and/or the antibody heavy chain constant domain is derived from a human IgGl, lgG2, lgG3, or lgG4 heavy chain constant région, particularly from a human IgG4 heavy chain constant région. “Derived from” means some punctual mutations by amino acid substitutions such as IgG4 (S228P) or IgGl(E333A). These mutations well known from the skilled person in the art, generally modify some parent chain properties. For example, they lead to less immunogenicity compare to the parental antibody or abrogate FcyReceptor binding or avoid dimerization of the monomer antibody or stabilize the dimerization rendering antibodies better for human therapeutical uses. The antibody of the invention derived from parental heavy and light chain constant domain comprises or consists of the sequence set forth in SEQ ID No: 26, SEQ ID No: 27 or in SEQ ID No: 28 or a fragment thereof, respectively. More particularly the antibody light chain constant domain consisting of the sequence set forth in SEQ ID No: 27. In particular, the antibody heavy chain constant domain comprises or consists of the sequence set forth in SEQ ID No: 26 or a fragment thereof. More particularly the antibody heavy chain constant domain consists of the sequence set forth in SEQ IDNo: 26.

[76] Provided herein are also isolated nucleic acid molécules encoding a polypeptide according to the invention, or an antibody or antigen-binding fragment thereof provided herein. Particularly, said nucleic acid molécules encode the 1 ight chain variable domain or the light chain of an antibody provided herein, in combination with isolated nucleic acid molécules encoding the heavy chain of an antibody provided herein, according to any of the définitions provided herein. In particular, provided herein is an isolated nucleic acid molécule encoding a polypeptide comprising or consisting of a sequence selected from the group consisting of SEQ ID No : 9; SEQ ID No : 10; SEQ ID No : 11; SEQ ID No : 12. In particular, the isolated nucleic acid molécule according to the invention comprises or consists of a sequence selected from the group consisting of SEQ ID NO : 15, SEQ ID NO : 16, SEQ ID NO :17 or SEQ ID NO :I8. Particularly, said isolated nucleic acid molécule is provided in combination with an isolated nucleic acid molécule encoding a heavy chain comprising the three CDRs consisting of the sequences set forth in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3, in particular encoding a heavy chain variable domain consisting of the sequence set forth in SEQ ID No: 7, more particularly the isolated nucleic acid molécule consisting of the sequence set forth in SEQ ID No: 13. In a preferred embodiment, a combination of isolated nucleic acid molécules encoding an antibody or antigen-binding fragment thereof is provided, said combination comprising or consisting of a first isolated nucleic acid molécule comprising or consisting of a sequence selected from the group consisting of SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17 and SEQ ID No: 18; and a second isolated nucleic acid molécule comprising or consisting of the sequence of SEQ ID No: 13. In another preferred embodiment, a combination of isolated nucleic acid molécules encoding an antibody or antigen-binding fragment thereof is provided, said combination comprising or consisting of a first isolated nucleic acid molécule comprising or consisting of the sequence SEQ ID No: 18; and a second isolated nucleic acid molécule comprising or consisting of the sequence of SEQ ID No: 13.

[77] Provided herein are also polynucleotides encoding the polypeptide sequence of a complété light chain, comprising both the variable and constant domain, i.e. in particular polynucleotides encoding of one ofthe sequences of SEQ ID No: 9 to 12 and the sequence of SEQ ID No:27 or 28, in particular polynucleotides comprising or consisting of one of the sequences SEQ ID No: 15 to 18 concatenated with SEQ ID No:30 or SEQ ID No:31. Such polynucleotides are provided in particular in combination with a polynucleotide encoding the polypeptide sequence of a complété heavy chain, comprising both the variable and constant domain, i.e. in particular polynucleotides encoding SEQ ID No: 7 and the sequence of SEQ ID No:26, in particular polynucleotides comprising or consisting of SEQ ID No: 13 concatenated with SEQ ID No:29.

[78] In particular, the isolated nucleic acid molécules provided herein may advantageously comprise, besides a sequence encoding a light chain and optionally a heavy chain of an antibody provided herein, upstream from the sequence encoding said antibody chains, a sequence encoding a signal peptide allowing sécrétion of said chains when expressed in a production cell. They may also comprise one or more sequence(s) encoding one or more marker peptide(s) for detecting, and/or facilitating the purification of, said chains.

[79] Provided herein is also a vector for the cloning and/or for the expression of a nucleic acid molécule provided herein. In particular, said provided vector is a plasmid suitable for cloning and/or expressing in manunalian cells, which comprises régulation sequences for transcription and expression. Accordingly, provided herein is a vector comprising a polynucleotide as disclosed above, in particular a polynucleotide as disclosed in paragraphs [76] to [78].

[80] Further, provided herein are cells or cell lines recombined with a nucleic acid molécule as above, in particular a vector, especially a mammalian or an avian cell or cell line, in particular as detailed in Figure 2. For example Chinese Hamster Ovary Cells, genetically modified to reduce global fucosylation. Indeed, antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cellmediated cytotoxicity (ADCC) (von Horsten et al., 2010). Another example is the EB66 cell line which naturally has low fucosylation properties (Olivier et al., 2010). Antibodies may also be produced in cells transiently transfected with a nucleic aid molécule as above, in particular a vector, in particular COS cells, in particular as detailed in Example 2.

[81] Provided herein are also methods for the production of a polypeptide provided herein, in particular an antibody light chain and/or a monoclonal antibody, said methods comprising the step of expressing said polypeptide, antibody light chain or monoclonal antibody (or the light and optionally the heavy chain of a monoclonal antibody) in cells comprising a nucleic acid molécule encoding said polypeptide, in conditions enabling the recovery of said polypeptide and the step of recovering said polypeptide. In particular, the antibodies or their fragment are prepared in cells that présent low fucosylation properties, such as EB66 avian cells.

[82] Provided herein is also a pharmaceutical composition comprising a polypeptide (in particular an antibody light chain variable domain or an antibody light chain) and/or an antibody or antigen-binding fragment or antibody mimetic molécule thereof and/or an isolated nucleic acid molécule as defined above, and a pharmaceutical vehicle. Said pharmaceutical composition can optionally further comprise a different active ingrédient. Said composition is provided in particular in a formulation suitable for systemic administration, or for local administration. In particular, provided herein are compositions suitable for local administration, in particular for intramuscular or subcutaneous injection, for injection using a device such as an autoinjector (or injector pen), for transdermal administration, in particular using a transdermal patch, for transmucosal administration, in particular intranasal or rectal administration. In particular, provided herein are compositions suitable for local administration to the gastrointestinal (Gl) tract, in particular through oral administration, in particular for the treatment of intestinal disease such as Crohn’s disease or UC, in particular compositions suitable for delivery to the colon. In particular, provided herein are compositions suitable for systemic administration, in particular for parentéral or enterai administration, in particular for intravenous injection or oral administration. As the skilled person is aware, enteric administration may be either a local administration to the Gl tract, or a systemic administration. Wherever such pharmaceutical compositions or their uses are provided herein, it must be understood that the administration vehicle and their uses may also be provided, e.g. when an injectable pharmaceutical composition is provided explicitly, it must be understood that the composition is provided as such as well as in a device or in combination with a device allowing administration and/or injection of said composition (“delivery device”). Examples of delivery devices include but are not limited to autoinjectors, in particular multichamber syringes, and transdermal patchs. Accordingly, provided herein are a delivery device, in particular an autoinjector, a pump, or a transdermal patch comprising said composition, and uses thereof as provided below in relation to the pharmaceutical compositions. Also provided herein are a kit comprising a pharmaceutical composition and a local delivery device, in particular a sub-cutaneous, enteric or oral delivery device, in particular a pre-filled syringe or a needle free device, containing said composition and/or suitable for the administration of said composition, and uses thereof as provided below in relation to the pharmaceutical compositions. The pharmaceutical composition is provided in any suitable form for administration, including as a solution, in particular a stérile aqueous solution, as a suspension, as a solid, in particular a lyophilized solid, in particular for adsorbtion on (or adsorbed on) a patch and/or for resuspension and adminsitration as a solution, as a pill, tablet or other solid form suitable for oral administration, in particular with delayed or extended release, as nanoparticles, e.g. a composition comprising nanoparticles with the polypeptide adsorbed on the surface, or within said nanoparticles, etc. The form of the pharmaceutical composition and optionally the nature of the delivery device may be suitable for delivery of the active ingrédient as provided herein systemically or locally, i.e. may be suitable for active ingrédient to reach the targeted cells, tissues, organs, in an active State. Particularly, the form of the pharmaceutical composition and optionally the nature of the delivery device may be suitable for delivery to the Gl tract, in particular to the colon. The pharmaceutical composition is said to comprise a pharmaceutically acceptable carrier; however, pharmaceutical compositions are also provided without a carrier for similar uses and purposes, when such a carrier is not required for pharmaceutical use, e.g. if the product is administered as a pure lyophilized solid. In particular, the administration is performed according to any suitable means as described herein for intenstinal release especially for intestinal delayed release.

[83] Provided herein is also a composition comprising as an active ingrédient, a polypeptide (in particular an antibody light chain variable domain or antibody light chain) and/or an antibody (in particular monoclonal antibody), antigen-binding fragment or antibody mimetic molécule thereof as defined above, or a pharmaceutical composition as defined above, in a formulation suitable for controlling human CD127 positive cells survival or expansion, in particular human CD127 positive effector cells, especially CD127+ memory T cells survival or expansion, especially memory T cells which are both CD127+ and CD8+, or which are both CD 127+ and CD4+ cells, when administered to a human patient. In particular, said composition comprising the antibody (or other agent as above) as an active ingrédient is in a formulation suitable for controlling the différentiation and / or maturation of dendritic cells when administered to a patient.

[84] The inventors hâve surprisingly shown that a single injection of the antibody provided herein allows for sustained effect, and the inflammatory response is still signifîcantly reduced in animais treated by a single injection ofthe provided antibodies as long as 14 months, and even as long as 18 months following said single injection. The administration, and preferably a single administration, of the polypeptide, in particular antibody light chain and more particularly the antibody, antigen-binding fragment or antibody mimetic molécule thereof provided herein, preferably has a fast and/or a longlasting effect on effector memory T cells. A fast effect is observed preferably within a week, and more preferably within 48 hours, and even more preferably within a day of the administration of said polypeptide, antibody chain or antibody. A long-lasting effect is observed preferably at least 12 months, and more preferably at least 14 months after the most recent (and, preferably, the single) administration of said polypeptide, antibody chain or antibody. Such an effet is in particular réversible (in particular T cell response may be restored by new vaccination). Such an effect is preferably antigen and/or responsespecific and ïs preferably not associated with measurable lymphodepletion (i.e. the global number of lymphocytes is not reduced in the subject, as measurey by common methods). Such an effect may otherwise be defined as a clonal délétion of memory T cells, or as a réversible, antigen spécifie délétion of immune T memory. An effect of such adminsitration on effector memory T cells may be assessed by comparing levels of IFN-γ secreting cells (measured e.g. by ELISPOT) in response to an antigen, e.g. tuberculine, in vaccinated subjects (in particular in baboons) which were later treated with the polypeptide, antibody chain or monoclonal antibody provided herein, and in untreated vaccinated subjects: an effect is observed if the measured level of antigen-specific T cells is signifîcantly lower (preferably at least 10 %, more preferably at least 40 % lower) in the treated subjects, at the relevant time point after treatment. Such an effect may also be measured by otherwise assessing inflammatory response to a given antigen, e.g. a local, in particular dermal, inflammatory réaction in response to an locally administered antigen. Alternatively, an MHC tetramer assay may be used. Methods to test for such effect are known to the skilled person and are illustrated herein in particular in Example 6.

[85] In addition, the inventors hâve suprisingly shown that the antibody provided herein may hâve a fast effect, in particular on T-cell activation, in particular on release of cytokines by T-cells, in particular in inflammatory tissue. Such an effect is in particular observed within a week, and preferably within 48 and even more preferably within 24 hours of administration of the antibody, and in particular is observed as a réduction in the IFNy production (or release). Such effect is in particular observed locally, i.e. at the site of administration of the antibody or at the site of delivery. In particular, the antibody provided herein has a fast effect, in particular a fast local effect, in particular on release of cytokines by T-cells, in particular has an effect observed within 24 hours of administration. When the antibody is administered for local delivery but not directly at the site of intended delivery, the antibody may hâve fast effect as above at the site of delivery, within the same time periods from delivery rather than from administration, which may or may not be delayed from administration as the skilled person is aware.

[86] A composition provided herein may further comprise an additional compound having a therapeutic immunomodulator effect, in particular on cells involved in allergy or autoimmunity. For illustration purposes, exemplary immunomodulators of interest are other monoclonal antibodies targeting T cells, such as anti-CD3, anti-lCOS or anti-CD28 antibodies or recombinant proteins or antibodies targeting accessory cells such as CTLA41g or anti-CD40 antibodies.

[87] The polypeptide, antibody, antigen-binding fragment or antibody mimetic molécule thereof, isolated nucleic acid molécule, cell and/or composition provided herein may be provided in a combination product, comprising additional products, in particular an agent with a therapeutic immunomodulator effect as above, in particular intended for simultaneously separately or sequentially administration. Provided herein is a combination product comprising a polypeptide (in particular an antibody light chain variable domain or antibody light chain) and/or an antibody, antigen-binding fragment or antibody mimetic molécule thereof, and/or an isolated nucleic acid molécule, vector, cell or cell line, and/or a pharmaceutical composition as defined above and optionally further comprising:

- an agent with a therapeutic immunomodulator effect, in particular intended for administration in combination with e.g. the antibody provided herein, in particular wherein said administration is either simultaneous or separated in time, and/or wherein said administration is through the same or a different route; and/or — a device for administration of the product.

[88] A polypeptide, in particular an antibody light chain and/or an antibody, in particular a monoclonal antibody and/or an antigen-binding fragment or antibody mimetic molécule and/or a nucleic acid, vector, cell or cell line and/or a pharmaceutical composition or a composition as defined above are in particular provided for use in a human patient for treating pathologies or pathologie conditions pathologie conditions influenced by immune responses, especially by memory T cells responses. Such conditions or pathologies comprise those induced by transplant rejection, autoimmune diseases, allergie diseases, respiratory diseases, chronic viral infections, chronic inflammatory disease, in particular chronic intestinal inflammatory disease, lymphoma, leukemia or other cancer diseases including those resulting from solid tumors when these pathologies are associated with CD127 positive cells as well as the IL-7 signalling pathway, in particular where an increase in the maturation of dendritic cells must be avoided. Accordingly, the inventors show that the use of said agents may be contemplated for the treatment of particular allergie skin disorders, inflammatory bowel disease (IBD), in particular Crohn’s disease (CD) or ulcerative colitis (UC), or Primary Sjôgren Syndrome, or Systemic Lupus Erythematosus, or Systemic Sclerosis or multiple sclerosis, or type 1 diabètes or acute lymphoblastic leukemia (e.g. T-ALL) or Hodgkin lymphoma, or breast cancer associated with CD127+ cells, rénal cancer, bladder cancer, lung cancer, pancreatic cancer, or for the treatment of a T cell cutaneous lymphoma, such as Sezary lymphoma, or for the treatment of the acute lymphoblastoid leukemia with gain-of-function mutation of the IL-7-R/TSLP pathway or for the treatment of transplant rejection and/or of patients in need of transplantation and/or about to undergo transplantation and/or in having undergone transplantion. The treatment of inflammatory bowel disease (IBD), in particular Crohn’s disease (CD) or ulcerative colitis (UC), or Primary Sjôgren Syndrome, or Systemic Lupus Erythematosus, or Systemic Sclerosis or multiple sclerosis, or type I diabètes is contemplated in preferred embodiments. In particular, the invention relates to the polypeptide, or the antibody or antigen-binding fragment or antibody mimetic molécule thereof, or the isolated nucleic acid molécule, or the pharmaceutical composition according to the invention for use as a médicament, more particularly for use in the prévention or treatment of organ or tissue transplant rejection or of a disease selected from the group consisting of autoimmune diseases, particularly rheumatoid arthritis, systemic sclerosis, multiple sclerosis, type 1 diabètes, autoimmune thyroiditis, systemic lupus erythematosus, primary sjôgren syndrome, and inflammatory diseases, particularly inflammatory bowel disease (IBD), more particularly Crohn’s disease and ulcerative colitis and encephalomyelitis and allergie diseases and cancer diseases and diseases related to transplantation and respiratory diseases, preferably by local administration.

[89] Provided herein are uses of the polypeptide, antibody light chain or antibody, antigen-binding fragment or antibody mimetic molécule thereof in the treatment of pathologie conditions involving the alteration of immune response in a human patient leading to dominant tolerogenic State or, to the contrary, lack of tolérance where control ofthe level ofthe immune response would be needed as well as destruction of malignant CD127-positive cells.

[90] By “treatment or “therapeutic treatment”, it is meant that the performed steps of administration resuit in improving the clinical condition of an animal or a human patient in need thereof, who suffers from disorder(s) associated with the 1L-7 pathway, i.e. involvingthe activation orproliferation of CD127 positive cells. Such treatment aims at improving the clinical status of the animal or human patient, by eliminating or alleviating the symptoms associated with the disorder(s) related to the IL-7 pathway, i.e. involving the activation or prolifération of CD 127 positive cells. Preferabaly, the treatment provided herein enables restoring to health. Preferably, said treatment does not hâve undesired négative effects due to increased maturation of immune cells, in particular of dendritic cells.

[91] In particular aspects of the treatment of patients, the polyeptide, antibody, antigen-binding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition is provided, intended and/or suitable for use to deplete CD127-positive cells while preserving CD127-negative cells.

[92] In particular aspects of the treatment of patients, the use of the polyepptide, antibody, antigenbinding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition is provided, intended and/or suitable for use to prevent différentiation and / or expansion and / or maturation of CD127-positive cells, in particular différentiation, expansion, or maturation induced by IL-7 and / or TSLP, while having little or no direct effect on CD127-negative cells.

[93] In particular aspects of the treatment of patients, the use of the polyepptide, antibody, antigenbinding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition is provided, intended and/or suitable for use to eliminate / neutralize naïve and memory T cells by interfering with lL-7-induced signaling, while preserving Treg cells.

[94] In particular aspects of the treatment of patients, the use of the polypeptide, antibody, antigenbinding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition is provided, intended and/or suitable for use to deplete subpopulations of lymphocytes, or other cell populations expressing CD127 (including normal or pathologie T and B lymphocytes, NK cells, dendritic cells and other cell types including épithélial cells) as a resuit of cytotoxic action of the antibodies, possibly but not exclusively through ADCC (Antibody-Dependent Cellular Cytotoxicity) and optionally through CDC (Complement-Dependent Cytotoxicity).

[95] Also provided herein is a polypeptide, in particular an antibody light chain and/or an antibody, antigen-binding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition as defined above, for use as active ingrédient in a combination or add-on therapeutic regimen in a patient in need thereof. Also provided is the use of a polypeptide, in particular an antibody light chain and/or an antibody, antigen-binding fragment, antibody mimetic molécule, polynucleotide, cell or cell line, or composition as defined above as a therapeutically active ingrédient in a combination or in an add-on therapeutic regimen in a patient in need thereof.

[96] In the aspects above, the contemplated uses are also applicable to nucleic acids, vectors, cells, cell Unes, compositions and pharmaceutical compositions provided hereinabove.

[97] Provided herein are the means and products above intended and/or suitable for use in pathologies such as those induced by transplant rejection, autoimmune diseases, allergie diseases, respiratory diseases, chronic viral infections, chronic inflammatoiy disease, in particular chronic intestinal inflammatory disease, lymphoma, leukemia or other cancer diseases including those resulting from solid tumors when these pathologies are associated with CD127 positive cells as well as the IL-7 signalling pathway, in particular where an increase in the maturation of dendritic cells must be avoided. Accordingly, said means and products are particularly intended and/or suitable for the treatment of particular allergie skin disorders, inflammatory bowel disease (IBD), in particular Crohn’s disease (CD) or ulcerative colitis (UC), or acute lymphoblastic leukemia (e.g. T-ALL) or Hodgkin lymphoma, or breast cancer associated with CD 127+ cells, rénal cancer, bladder cancer, lung cancer, pancreatic cancer, or for the treatment of a T cell cutaneous lymphoma, such as Sezary lymphoma, or for the treatment of the acute lymphoblastoid leukemia with gain-of-function mutation of the IL-7-R/TSLP pathway or for the treatment of transplant rejection and/or of patients in need of transplantation and/or about to undeigo transplantation and/or in having undergone transplantion.

[98] In particular, provided herein are the use of a polypeptide, in particular an antibody light chain and/or an antibody, antigen-binding fragment, antibody mimetic molécule, a nucleic acid, cell, cell line or composition in a human patient for the treatment of conditions and/or pathologies induced by transplant rejection, autoimmune diseases, allergie diseases, respiratory diseases, chronic viral infections, chronic inflammatory disease, in particular chronic intestinal inflammatory disease, lymphoma, leukemia or other cancer diseases including those resulting from solid tumors when these pathologies are associated with CD127 positive cells as well as the 1L7 signalling pathway, in particular where an increase in the maturation of dendritic cells must be avoided. Accordingly, said products are particularly for the treatment of particular allergie skin disorders, inflammatory bowel disease (IBD), in particular Crohn’s disease (CD) or ulcerative colitis (UC), or acute lymphoblastic leukemia (e.g. TALL) or Hodgkin lymphoma, or breast cancer associated with CD 127+ cells, rénal cancer, bladder cancer, lung cancer, pancreatic cancer, or for the treatment of a T cell cutaneous lymphoma, such as Sezary lymphoma, or for the treatment of the acute lymphoblastoid leukemia with gain-of-function mutation of the IL7-R/TSLP pathway or for the treatment of transplant rejection and/orof patients in need of transplantation and/or about to undergo transplantation and/or in having undergone transplantionan autoimmune disease or an allergie disease or for the treatment of leukemia such as acute lymphoblastic leukemia or for the treatment of lymphoma, or for the treatment of cancer disease, or for the treatment of a chronic viral infection, or for the treatment of inflammatoiy diseases, in particular IBD, particularly CD or UC, or for the treatment of respiratory diseases, or for the prévention and/or treatment of symptoms related to a transplantation.

[99] In particular, provided herein is a method of treatment comprising the administration of a polypeptide (in particular an antibody light chain variable domain or an antibody light chain) and/or an antibody, antigen-binding fragment, antibody mimetic molécule, or an isolated nucleic acid molécule, cell, and/or cell line or a composition and/or pharmaceutical composition as defïned above in a human patient for the treatment of conditions and/or pathologies induced by transplant rejection, autoimmune diseases, allergie diseases, respiratory diseases, chronic viral infections, chronic inflammatory disease, in particular chronic intestinal inflammatory disease, lymphoma, leukemia or other cancer diseases including those resulting from solid tumors when these pathologies are associated with CD127 positive cells as well as the IL-7 signalling pathway, in particular where an increase in the maturation of dendritic cells must be avoided. Accordingly, said methods are particularly for the treatment of particular allergie skin disorders, inflammatory bowel disease (IBD), in particular Crohn’s disease (CD) or ulcerative colitis (UC), or acute lymphoblastic leukemia (e.g. T-ALL) or Hodgkin lymphoma, or breast cancer associated with CD 127+ cells, rénal cancer, bladder cancer, lung cancer, pancreatic cancer, or for the treatment of a T cell cutaneous lymphoma, such as Sezary lymphoma, or for the treatment of the acute lymphoblastoid leukemia with gain-of-function mutation of the 1L7-R/TSLP pathway or for the treatment of transplant rejection and/orof patients in need of transplantation and/or about to undergo transplantation and/or in having undergone transplantionan autoimmune disease or an allergie disease or for the treatment of leukemia such as acute lymphoblastic leukemia or for the treatment of lymphoma, or for the treatment of cancer disease, or for the treatment of a chronic viral infection, or for the treatment of inflammatory diseases, in particular IBD, particularly CD or UC, or for the treatment of respiratory diseases, or for the prévention and/or treatment of symptoms related to a transplantation.

[100] The inventors hâve further shown that in patients with UC, levels of 1L7, CD127 and/or TSLPR mRNA allow to predict response to conventional immunosuppressive treatments: résponders (i.e. patients which exhibit marked symptom réduction in response to treatment) hâve lower levels of IL-7 and CD 127 and higher levels of TLSPRthan non-responders. Accordingly, provided herein are in vivo methods to assess the likelihood of response to immunosuppressive treatments in patients, in particular human patients, having ulcerative colitis (UC) comprising measuring in a sample obtained from said patient the level of IL7, CD 127 and/or TLSPR mRNA or protein, and concluding that the likelihood of response is increased compared to a reference group when the measured level of IL7 and/or CD127 is lower than the average level in said group and/or when the measured level of TSLPR is higher than the average level in said group. Methods to perform the required measurements are known to the skilled person and may involve the use of an antibody against CD127 in particular for the measurement of the protein expression levels of CD127. The polypeptide, particularly the antibody light chain and/or the monoclonal antibody provided hereinabove is provided in particular for use in such methods and such methods are provided in particular using such a polypeptide, antibody light chain and/or the monoclonal antibody.

[101] In particular, provided herein is a method of selecting a compound from the group consisting of an antibody, an antigen-binding fragment thereof and an antibody mimetic molécule, the method comprising at least one of the following steps:

i. testing the binding of the compound to CD 127, in particular to human CD 127, in particular to an epitope sequence from domain DI and/or from the site 2b of domain D2 of CD 127.

An epitope sequence from domain DI and/or from the site 2b of domain D2 may be any one of the epitope sequences described therein, particularly as disclosed in paragraphs [016], particularly an epitope sequence selected from the group consisting of SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36 or SEQ ID No. 96, and more particularly SEQ ID No 34, SEQ ID No. 35 and SEQ ID No. 96. In a particular embodiment of the invention, the binding test may encompass testing the binding of the compound to an epitope sequence selected from the group consisting of SEQ ID No: 34, SEQ ID No. 35 and SEQ ID No. 96, and testing the binding of the compound to an epitope sequence selected from the group consisting of SEQ ID No. 42 and SEQ ID No. 43. The binding capacity of the compound may be tested by any one of the methods know in the art, in particular the Blitz method, known to the skilled person and illustrated in particular in Figure 3, Example 3 and Example 4 herein and in p. 14, Figures 3,4 and 6 and the respective legends, and Examples 1,2, 6,7 of WO 2015/189302., and/or by the method of example 10 disclosed herein; and/or ii. testing the inhibition of IL7-R signalling induced by IL-7, in particular STAT5 phosphorylation, in presence of the compound. The inhibition induced by 1L7 in presence of the compound may be tested by the method disclosed in example 8 and/or example 11 herein; and/or iii. testing the activation of the phosphatidylinositol 3-kinase in presence of the compound. The activation of the phosphatidylinositol 3-kinase in presence of the compound may be tested according to the method disclosed in example 8 and/or example 11 herein; and/or iv. testing the activation of the ERK signalling pathway in presence of the compound. The activation of the ERK signalling pathway in presence of the compound may be tested according to the method disclosed in example 8 and/or example 11 herein;

v. testing the binding capacity of the compound to at least one beta sheet of the site 2b of domain D2 of CD127, in particular at least to the third beta sheet of site 2b, defined as being the nucleo acids of SEQ ID No. 34, and/or to at least one amino acid sequence selected from the group of SEQ ID No. 35 and SEQ ID No. 96. The binding capacity of the compound may be tested by any one of the methods know in the art, in particular the Blitz method, known to the skilled person and illustrated in particular in Figure 3, Example 3 and Example 4 herein and in p. 14, Figures 3,4 and 6 and the respective legends, and Examples 1,2, 6,7 of WO 2015/189302., and/or by the method of example 10 disclosed herein.

The method may further comprise any one of the following optional steps, or at least one of the following optional steps:

vi. testing the binding of CD 127, in particular human CD 127, to the yc common chain of cytokine receptors in the presence of the compound. The binding of CD127 to the yc common chain of cytokine receptors in the presence of the compound may be assessed in particular by co-immunoprecipitatîon methods, well known to the skilled person for testing the interaction of proteins and illustrated e.g. in WO2015/189302 in Example 21. In particular, cells may be incubated in the presence or absence of the tested compound, then solubilized in conditions allowing for the préservation of protein complexes, and the resulting lysate may be subjected to an anti-CD127 immunoprécipitation and the presence of yc in the CD127-containing immunoprecipitated complex assessed by western blotting using anti-yc antibodies (conversely, the immunoprécipitation may be performed using antiyc antibodies and the presence of CD127 assessed using anti-CD127 antibodies); and/or vii. testing the întemalization of CD127, in particular human CD127, and/or lL7-induced intemalization of CD127 in presence of the compound. The întemalization of CD127, herein defined in paragraphs [51] to [53], may be further defined and/or tested as set forth in WO2015/189302 in particluar in paragraphs [59]-[63] at pages 19-20 and in Figure 16 and example 5; and/or viii. testing the maturation of dendritic cells induced by TSLP in presence of the compound. The maturation of dentritic cells induced by TSLP is defined in paragraphs [47] and [48] herein. The means to measure such effect are known to the skilled person and are disclosed in particular in WO 2015/189302 at pages 16-17 and in Example 9 thereof. In particular, the means to measure such effect comprise measure of the expression of CD40 between cells stimulated with the compound and cells stimulated with TSLP alone (without the compound).

In a particular embodiment of the method, the compound which specifically binds to CD 127, in particular human CD127, which is an antagonist of1L7-R signalling induced by 1L7, which does not induce the activation ofthe phosphatidylinositol 3-kinase and/or the ERK signalling pathway is selected. In a more particular embodiment of the method, the compound which specifically bind to CD 127, in particular human CD 127, which is an antagonist of 1L7-R signalling induced by IL7, and which does not induce the activation ofthe phosphatidylinositol 3-kinase and does not induce the activation ofthe ERK signalling pathway is selected.

[102] In particular, provided herein is a method for producing an antibody or an antigen-binding fragment thereof ofthe invention, which is raised against CD 127, possibly raised from an immunization of a non-human animal, such as rats of the LOU/C Igkla strain available at the university of Louvain, Belgium). Immunization can be carried out using a fragment ofthe amino acid sequence of SEQ ID No.

22, and in particular a fragment of SEQ ID No. 22 comprising an epitope sequence as defined herein, and in particular SEQ ID No. 35 and/or SEQ ID No. 96, as an immunogen. Hybridoma may be obtained by fusing spleen mononuclear cells with the LOU rat immunocytoma 1R983F. Hybridoma may be screened according to the capacity of the secreted monoclonal antibodies to bind to an amino acid sequence selected from the group consisting ofSEQ ID No: 22, SEQ ID No. 35 and SEQ ID No. 96, in particular SEQ ID No: 35 and/or SEQ ID No: 96. Therefore, the invention also encompasses an immunogen compound, said immunogen compound being a fragment of the amino acid sequence of SEQ ID No. 22, in particular a fragment comprising at least one amino acid sequence selected from the group ofSEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 96, in particular SEQ No. 34, SEQ ID No. 35 and SEQ ID No. 96, more particularly SEQ ID No. 96. In a particular embodiment of the invention, the immunogen compound is a linear peptide, in particular a linear peptide comprisingthe amino acid sequence ofSEQ ID No. 96, more particularly, a linear peptide comprising or consisting of, preferably consiting of, the amino acid sequence ofSEQ IDNo. 35.

Brief description of the drawings

Figure 1. Amino acid sequences of the antibodies provided herein

Panel A. Sequence of the heavy chain (VH). The CDRs are in bold characters.

Panel B. Sequence of the humanized light chains (LH) derived from N13B2. The CDRs are in bold characters. N13B2-VL3 comprises orignal framework residues of N13B2-h3 in positions 48 and 87 (underlined); N13B2-VL4-V48L and N13B2-VL5-F87Y comprise the corresponding humanized framework residue andN13B2-VL6-V48L-F87Y comprises both humanized framework residues.

Figure 2. Production of antibodies - stable transfection

Three different production batches were performed for each of the mentioned antibodies in CHO-M cells according common methods, using commercially available serum-free media, and titers obtained in typical experiments, measured by an ELISA assay, are reported here. The horizontal axis represents the culture time, in days, while the vertical axis represents the obtained antibody titers, in pg/mL.

Figure 3. Binding to CD127

Binding of the indicated antibodies to recombinant CD127 was assayed by ELISA according to the method detailed in Example 3. The horizontal axis represents antibody concentration in ng/mL, and the vertical axis represents optical density at 450 nm, in arbitrary units.

Figure 4. Inhibition of STAT5 phosphorylation

The inhibition of STAT5 phosphorylation by the indicated antibodies was assayed by cytofluorometry according to the method detailed in Example 5. The percentage of CD3+ cells stained with pSTAT5 antibodies is reported in panel A (horizontal axis: antibody concentration in ng/mL) and the mean fluorescence intensity (in arbitrary units) of pSTAT5 signal for CD3+ cells is reported in panel B.

Figure 5. Effect on memory T cells

Panel A. DTH response (Area under curve of erythema curves) in baboon afterN13B2 injection.

Delayed-type hypersensitivity in response to a tuberculin challenge was assayed in vaccinated baboons, after administration ofN13B2 (n=7 baboons) or excipient (n=4 baboons), by measuring dermal reaction according to the method detailed in Example 6. The vertical axis represents area under the erythema curve, in arbitrary units. Values are reported for intradermal reactions performed at the given time points, indicated in days on the horizontal axis, before or after the administration of N13B2 or excipient (administered at day 0). “Post-BCG” corresponds to dermal reaction results performed after a new vaccination with BCG (nd = not determined).

The excipient control was not determined at days 150 and 180 and “post-BCG” (nd).

Panel B.

DTH response (Area under curve of erythema curves) in baboon after humanized N13B2 injection.

Delayed-type hypersensitiviy in response to a tuberculin challenge was assayed in vaccinated baboons, after administration of humanized N13B2 (AA892BB, 32257, V915GQ, 33874) or buffer, by measuring dermal reaction according to the method detailed in Example 6. The vertical axis represents area under the erythema curve, in arbitrary units. Values are reported for intradermal reactions performed before the administration of humanized N13B2 or buffer (“1DR1”, first bar from left for each baboon), 4 hours after administration of humanizedNI3B2 or buffer (“IDR2”, second bar from left), one, two and three months (“IDR3-5”, third to fifth bar from left) and four months (“IDR6”, sixth bar from left, for V915GA only) after administration of humanized N13B2 or buffer and after a new vaccination with BCG (“1DR7”, last bar from left).

Panel C.

IFNy EL1SPOT performed on blood PBMC in BCG-vaccinated baboon challenged with tuberculin and treated with humanized NI 3B2.

AG-specific T cell frequency after tuberculin challenge was assayed in vaccinated baboons after administration of humanized NI 3B2 (AA892BB, 32257, V915GQ, 33874) or buffer, by IFNyELISPOT assay according to the method detailed in Example 6. The vertical axis represents spot frequency for 100.000 cells. Values are reported for Elispot without antigen (W/o Ag, left bars) or with tuberculin antigen (right bars) performed before the administration of humanized N13B2 or buffer (first bar from left for each group of bars), 4 days after administration of humanized N13B2 or buffer (“IDR2”), one, two and three months (“1DR3-5”) and four months (“1DR6”, for V915GA only) after administration of humanizedNI3B2 or buffer and after a new vaccination with BCG (last bar from left, dashed).

Figure 6. Expression of IL-7, CD127 and TSLP in IBD patients mRNA expression levels of IL-7, CD127 (soluble form of IL-7Ra) and TSLP (full-length) were measured according to the method detailed in Example 7 intissue samples from healthy control subjects (Non-IBD control), healthy and diseased (inflammed) colon biopsy samples from patients with active ulcerative colitis (UC) who did not respond (or no longer responded) to antiinflammatory treatment and in samples from UC patients with quiescent disease — i.e. were cured or in remission at the time of sampling (Responders). The vertical axis represents relative fluorescence units. The value is plotted for each sample in a given group, the horizontal bar representing the average values for the group and the error bars represent the standard déviation. dénotés a p-value < 0.05; “**”a p-value < 0.0T, “****” a p-value < 0.0001.

Figure 7. Inhibition of CD127 signalling pathways

The effect of various anti-human CD127 antibodies on the activation of signaling pathways was assessed by Western Blot as detailed in Example 8. The figure represents représentative results from 6 different donors. “No IL-7” corresponds to a sample which was not stimulated by IL-7. “C” corresponds to a control sample, stimulated with IL-7 in the absence of anti-CD127 antibody. Horizontal Unes left of the blot represent the migration of the indicated molecular weight marker. Arrows right of the blot indicate the migration of tyrosine-phosphorylated STAT5, tyrosine 199-phosphoiylated PI3-k p55, phosphorylated Akt, phosphorylated ERK 1/2, and, as a loading reference, GAPDH.

Figure 8. Smothering of T-cell cytokine release from UC biopsies

Panel A. IFNy production by UC biopsy samples grown ex-vivo. Panel B. IFNy production by CD biopsy samples grown ex-vivo.

In both panels, the samples were obtained and treated as detailed in Example 9. Each symbol represents one sample from a patient, cultured with IgG (“Ctrl Ab”) or with anti-CD 127 antibody (“alL-7Ra”). Connected symbols are paired samples from the same patient. ** p<0.01 with Wilcoxon matched pairs test. IFNy production was significantly inhibited by anti-IL7Ra mAb. Similar results were observed for CD biopsy samples.

Figure 9. Anti-human IL-7Ra mAbs and agonist signais

The effect of various anti-human CD127 antibodies on the activation of STAT5, P13K and ERK signaling pathways was assessed by Western Blot as detailed in Example 11. The figure 9A shows from one out of seven different donor cells in the absence of exogenous recombinant human IL7. Panel A. “medium” corresponds to a sample without any anti-human CD127 antibody. “No 1L7” means that the four samples were not stimulated with IL-7. Three samples were pretreated with lOpg/mL of one anti IL-7Ra mAb (N13B2-hVL6 or MD707-13-G4 or 1A11-G1 ).

Panel B. Quantification of p!3K and pERK corrected to GAPDH expression and normalized to medium control conditions (n=7 different donnors). The vertical axis represents the normalized expression to control. The value is plotted for each sample in a single group, the horizontal bar representing the average value for the group, and error bars representing the standard déviation.

Figure 10. Effect of Anti-human IL7-Ru mAbs on IL7 pathway activation

The quantification of phospho-STAT5 signal was corrected to GADPH expression and normalized to medium control conditions. PBMCs were pretreated with lOpg/mL of one anti-IL7-Ra mAb (N13B2hVL6 or MD707-13-G4 or 1A11) and then incubated for 10 min at 37 °C with 5ng/mL of human 1L7. Quantifications of phospho-STAT5 signal were corrected to GAPDH expression (n=7 different donors). The doted line represents the condition with medium alone without treatment. The value is plotted for each sample in a single group, the horizontal bar representing the average value for the group, and error bars representing the standard déviation. dénotés a p-value < 0.05 between indicated groups.

Figure 11. Dual agonist/antagonist anti-IL7-Ru mAbs induce transcriptional modifications

RNA sequences analysis ofhumanPBMCs (n=7) incubated for 3.5 hours (1) with 5ng/ml of human IL7, (2) without IL7, (3,4,5) with 5ng/mL of human IL7 and different anti-human IL7-Ra mAbs ((3): lOpg/mL N 13B2-hVL6; (4): 10pg/mL MD707-13-Ig4; (5): 10pg/mL 1 Al 1 ).

Panel A. Heatmap ofthe expression ofthe 93 most differentially expressed genes (False Discovery Rate (FDR) 5%, Fold change (FD) > 2) between IL7 stimulation and control conditions.

Panel B. Quantification of the médian profile of the three 1L7 induced clusters in 1L7 stimulated, control and 1L7 and anti-human IL7-Ra mAbs conditions.

Panel C. Venn diagram of RNA sequences analysis of human PBMCs (n=7) incubated without IL-7 for 3.5 hours with different anti-human IL-7Ra mAbs (10 pg/mL of N 13B2-hVL6, lOpg/mL MD707-13Ig4#I, or 10pg/mL 1AI1). Venn diagram ofthe 481 differentially expressed genes (FDR 5%, FC > 1.5) comparing anti-human IL-7Ra mAbs and medium control conditions. Circle size is proportional to the number of genes for each category.

Examples

Example 1. Humanization of light chains

The following heavy chain was used in ail experiments reported herein, unless provided otherwise

N13B2 humanised_VH, nucléotide sequence (SEQ ID No: 13):

CAGGTGCAGCTGGTCGAATCAGGGGGGGGACTGGTCAAACCCGGGGGCTCACTGCGTCTGTCATGTGCCG TCTCAGGCTTCACACTGAGCGACTACTATATGGCATGGATCCGACAGGCACCAGGCAAGGGACTGGAGTGG GTGTCTACTATTTCTGCCAGTGGCCTGAGGACCTACTATCCTGACAGTGTCAAGGGAAGGTTCACAATCTCAC GGGATAACGCTAAAAATTCCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAAGACACCGCTGTGTACTATr

GCGCTCGCCCACTGTCCGCACACTATGGCTTCAATTACTTTGATTATTGGGGGCAGGGTACCCTGGTGACAG TCTCCAGC

N13B2 humanised_VH, Amino-acid sequence (SEQ ID No: 7): see Figure 1A

The following optimized nucléotide sequences were used for the production of the antibody light chains (the amino acid sequences of which are provided in Figure IB):

N13B2-h3 (SEQ ID No: 14):

GAGATCGTCATGACGCAGTCCCCCGCAACGCTCTCCGTCTCCCCGGGGGAACGCGCGACC CTGTCGTGCAGGACCTCCGAGGACATCTACCAAGGCCTCGCGTGGTATCAGCAGAAGCCC GGCCAGGCCCCGCGGCTGTTGATCTACTCCGCGAACACCTTGCACATCGGCATCCCGGCG CGCTTCTCGGGGTCAGGGAGCGGCACCGAGTTCACCCTGACCATCTCGTCGCTCCAGAGC GAGGACTTCGCCGTGTACTACTGCCAGCAGTACTACGACTACCCCCTGGCGTTCGGGGGC GGGACCAAGGTGGAGATCAAG

N13B2hVL3 (SEQIDNo: 15):

GACATTCAGATGACCCAGTCCCCCTCGAGCCTGAGTGCGAGTGTGGGCGACCGCGTGACG ATCACCTGCCGGACGTCCGAGGATATCTACCAGGGCCTCGCCTGGTACCAGCAGAAGCCG GGCAAGGCCCCCAAACTGCTGGTCTACAGCGCGAACACCCTCCACATCGGCGTCCCCAGC CGGTTCAGCGGCTCCGGCTCGGGAACGGACTACACCCTCACGATCTCGTCCCTGCAGCCG GAAGACTTCGCCACCTACTTCTGCCAGCAGTATTACGACTACCCGCTGGCGTTCGGTGGC GGCACCAAGGTCGAGATCAAG

N13B2hVL4 (SEQ ID No: 16):

GACATTCAGATGACCCAGTCCCCCTCGAGCCTGAGTGCGAGTGTGGGCGACCGCGTGACG ATCACCTGCCGGACGTCCGAGGATATCTACCAGGGCCTCGCCTGGTACCAGCAGAAGCCG GGCAAGGCCCCCAAACTGCTGCTCTACAGCGCGAACACCCTCCACATCGGCGTCCCCAGC CGGTTCAGCGGCTCCGGCTCGGGAACGGACTACACCCTCACGATCTCGTCCCTGCAGCCG GAAGACTTCGCCACCTACTTCTGCCAGCAGTATTACGACTACCCGCTGGCGTTCGGTGGC GGCACCAAGGTCGAGATCAAG

N13B2hVL5 (SEQ ID No: 17):

GACATTCAGATGACCCAGTCCCCCTCGAGCCTGAGTGCGAGTGTGGGCGACCGCGTGACG ATCACCTGCCGGACGTCCGAGGATATCTACCAGGGCCTCGCCTGGTACCAGCAGAAGCCG GGCAAGGCCCCCAAACTGCTGGTCTACAGCGCGAACACCCTCCACATCGGCGTCCCCAGC CGGTTCAGCGGCTCCGGCTCGGGAACGGACTACACCCTCACGATCTCGTCCCTGCAGCCG GAAGACTTCGCCACCTACTACTGCCAGCAGTATTACGACTACCCGCTGGCGTTCGGTGGC GGCACCAAGGTCGAGATCAAG

N13B2hVL6 (SEQIDNo: 18):

GACATTCAGATGACCCAGTCCCCCTCGAGCCTGAGTGCGAGTGTGGGCGACCGCGTGACG ATCACCTGCCGGACGTCCGAGGATATCTACCAGGGCCTCGCCTGGTACCAGCAGAAGCCG GGCAAGGCCCCCAAACTGCTGCTCTACAGCGCGAACACCCTCCACATCGGCGTCCCCAGC CGGTTCAGCGGCTCCGGCTCGGGAACGGACTACACCCTCACGATCTCGTCCCTGCAGCCG GAAGACTTCGCCACCTACTACTGCCAGCAGTATTACGACTACCCGCTGGCGTTCGGTGGC GGCACCAAGGTCGAGATCAAG

Each VL sequence was obtained by gene synthesis, inserted in a cloning vector (pUC57) with BsiWI 5’ and 3’ extremities and the addition of a Kozak sequence (GCCACC) before the ATG. As expression vector, pFuseCLlg-hk expression plasmid (Invivogen) was used, containing the CLkappa constant domain of human IgGL

Each cloning plasmid (VL-pUC57-Genscript) was digested by BsiWI restriction enzyme to extract the VL insert (400 bp). The purified insert was ligated in the expression plasmid pFuseCLIg-hk linearized by BsiWI digestion and dephosphorylated. Positive clones, which hâve VL fragments inserted in the right orientation before human constant domains, were amplified and purified by Midiprep-endotoxin free (Macherey-Nagel) for transfection step. The heavy chain was cloned in a similar fashion, with the constant domain consisting ofSEQ ID No:26.

Example 2. Production of humanized light chains

For the tested humanized anti-CD127 antibodies, transfection and sélection of stable clones were made according to conventional methods. Three supematants for each antibody corresponding to different ratio oftransfection ofthe heavy chain (HC) and light chains (LC) wereprepared and tested: 2:1, 1:1.3, and 1:2 HC:LC. Titers obtained are reported in Figure 2, obtained following production in CHO cells seeded at 300'000 cells/mL; without antibiotics, according to conventional methods: the titer was assayed by ELISA on immobilized anti-human IgG (Fc) of the corresponding antibody and révélation was performed with a mouse anti-human kappa mAb plus peroxidase-labeled donkey anti-mous e antibodies and revealed by colorimetry at 450nm using TMB substrate.

Production ofN13B2-h3 and N13B2-hVL6 was also tested in transient transfection experiments. One day before transfection, COS cells were seeded at 100 000 cells/well in P12 plate with completed medium (DMEM SVF10% (Hyclone) +PS 1% + Glu 1%) and incubated at 37°C, 5%CO2. The day of transfection, COS cells were used at 50 to 90% confluence. They were washed with PBS and kept with 500μ1 in completed medium. 0.6 pg VH variant + 0.4pg VL variant were mixed in 200μ1 OptiMEM medium and 1 μΐ of Plus Reagent (Invitrogen) was added (incubation 15min at room-temperature). 3.5μ1 lipofectamine LTX (lnvitrogen)+100pl were added in the mix and incubated 25min at roomtemperature. The whole mix was deposited drop by drop on COS cells and incubated 48h at 37%,

5%CO2. After 48h, supematants were harvested and centrifuged (1500rpm 10min 4°C). Supematants were quantified with ELISA n°TH-MO-43. Activity assay was made with ELISA TH-MO-44.

Example 3. Binding to CD127 - Elisa

For sandwich ELISA, donkey anti-human IgG (Fc spécifie) antibody was coated at 1.2pg/ml on P96plate and purified antibodies were added to measure concentration in function of standard range. After incubation and washing, mouse anti-human light chain, kappa spécifie, (Effimune, clone NaM76-5F3) plus peroxidase-labeled donkey anti-mouse (Jackson Immunoresearch, reference 715-036-151) antibodies were added and revealed by conventional methods.

For activity ELISA assay, recombinant hCD127 (Sino Biologicals, Beijing, China; reference 10975H08H) was immobilized on plastic at 1 pg/ml and dilutions of anti-CD 127 antibody were added to measure binding. After incubation and washing, mouse anti-human light chain (kappa spécifie) plus peroxidase-labeled donkey anti-mouse antibodies were added and revealed by colorimetry at 450nm using TMB substrate by conventional methods.

The following ED50 values were obtained (concentration required to achieve 50 % of the maximum signal):

	ED50 (ng/ml)
N13B2-h3	16,8
N13B2-hVL3	15,1
N13B2-hVL4	12,6
N13B2-hVL5	14,8
N13B2-hVL6	9,5

Table 1. ED50 value (In ng/ml) for binding to CD127 ofthe antibodies

Stability ofthe antibodies was studies by incubating the antibodies for 7, 14, or 30 days at 4 °C, 25 °C and 42 °C. The binding of the antibodies to CD 127 was still excellent even after 30 days incubation.

Values of ED50 determined by ELISA after 30 days of incubation are reported in table 2.

	ED50 (ng/ml)
N13B2-hVL6 d7 at 4°C	55,31
N13B2-hVL6 d7 at 25°C	47,83
N13B2-hVL6 d7 at 42°C	56,16

N13B2-hVL6 d!4 at4°C	62,75
N13B2-hVL6 d!4 at 25°C	52,58
N13B2-hVL6 d!4 at 42°C	40,62
N13B2-hVL6 d30 at 4°C	46,98
N13B2-hVL6 d30 at 25°C	40,19
N13B2-hVL6 d30 at 42°C	58,69

Table 2. ED50 value (in ng/ml) for binding to CD127 ofthe antibodies, after30 days Incubation at the indicated température.

Example 4. Binding to CD127 - Blitz

This method was performed with a Blitz (Forte Bio, C22-2 No 61010-1).

Recombinant hCD127 (Sino Biologicals, Beijing, China; reference 10975-H08H) /recombinant protein (Sino Biological Cat: 11612-H08H) was immobilized at 50pg/ml by Fc fragment into anti-human IgG Fc (AHC) biosensor (Forte Bio, 18-5063) for 30 seconds. Then, anti-CD127 antibodies were added at 20pg/mL (saturating concentration) for an association period of 120 seconds, followed by a dissociation period of anti-CD127 antibody in kinetics buffer for 120 seconds. Data analysis was made with the Blitz pro 1.2 software, which calculated association constant (ka) and dissociation constant (kd) and determined the affinity constant KD (ka/kd). Results are reported in Table 3.

	Association (ka) (1/Ms)	.Dissociation (kdj (1/s)	Affinity (KD) (M)
N13B2-h3	l,15e6	2,67e-3	2,33e-9
N13B2-hVL3	l,27e6	4,47e-3	3,51e-9
N13B2-hVL4	l,04e6	2,81e-3	2,71e-9
N13B2-hVL5	l,01e6	2,66e-3	2,63e-9
N13B2-hVL6	l,05e6	2,66e-3	2,53e-9

Table 3. Affinity analysis by Blitz of anti-CD127 antibodies on human CD127 recombinant protein

Example 5. Inhibition of STAT5 phosphorylation

To test inhibition of IL7R in functional assay, antibody was incubated with human PBMC for 30min at 37°C, before stimulating with IL7 (AbD Serotec, ref PHP046) at O.lng/ml for 15min at 37°C. Reaction was stopped a 4°C, and washed with Perm Wash buffer before fixation with Cytofix/Cytoperm kit (BD Bioscience, ref 554722) for 15 min at 4°C. Cells were washed and stained with FITC-labelled anti-CD3 (BD Bioscience, ref 557694) for 30min at 4°C. Then, cells were permeabilized in incubating Perm

Buffer III (BD Bioscience, ref 558050) for 30min at 4°C. Aller vvashing with PBS BSA1% Azide 0.1%, cellq were stained with Alexa-647 labelled anti-pStat5 antibody (BD Bioscience, ref 612599) for 30min at room-temperature. Sam pies were analysed on BD Cantoll cytofluorometer. hPBMC-CD3+ with 1L7 induced phosphorylation of pStat5, whereas, without IL7, we hâve no phosphorylation. Results are reported in Figure 4 and the table below, displaying ED50, i.e. the concentration of the indicated antibody to reach 50% of the signal in this assay.

	IC50-MFI ; (ng/ml)
N13B2-h3	21,5
N13B2-hVL3	27,2
N13B2-hVL4	16,1
N13B2-hVL5	27,3
N13B2-hVL6	16,8

Table 4. Inhibition ofSTAT5 phosphorylation by antl-CD127 antibodies

This experiment confïrmed that modification of germline and modification of structural residues by humanized amino-acid did not change the biological activity of anti-CD 127 antibody. Ail tested variants (N13B2-h3, N13B2-hVL3, N13B2-hVL4, N13B2-hVL5, N13B2-hVL6) could inhibit Stat5 phosphorylation after 1L7 stimulation on hPBMC, like previously produced reference batches of NI3B2. Focusing on N13B2-hVL6 (the most humanized and the most optimized), we noticed that it was able to maintain its biological activity in inhibiting Stat5 phosphorylation, to the same extent than reference N13B2-h3. Moreover, this variant was very stable after 14 days of incubation at 42°C, 25°C or 4°C, it did not induce aggregate formation, and maintained its binding activity.

Example 6. Effect on memory T cells

Tuberculin-induced delayed-type hypersensitivity model

Baboons were immunized intradermally twice with a bacillus Calmette-Guerin vaccine (0.1 ml; 2—8 105 CFU; Sanofi Pasteur MSD, Lyon, France) in the upper région of the leg, 4 and 2 weeks before the delayed-type hypersensitivity (DTH) skin test. Intradermal reactions (1DR) were performed with intradermal injections of2000 or 1000 UI of Tuberculin Purified Protein Dérivative (PPD; Symbiotics Corporation, San Diego, CA). Saline (0.1 ml) was used as a négative control. Dermal responses at the injection sites were measured using a caliper square by at least two observers and were considered positive when > 4 mm in diameter. A second 1DR was performed after a three-week washout period and animais received one intravenous injection of 10 mg/Kg of N13B2 (n = 7) or lOmg/kg (V915GA, AA892BB, 32257, 33874) (n = 4) of humanized N13B2 or équivalent of volume excipient (n = 4). Additional IDR were performed evety month after the injection. After a washing period, some baboons (previously treated with N13B2) were immunized again intradermally twice with a BCG following by a new IDR. The mean of the reading was recorded and plotted for each time point. To compare multiple experimental conditions, eiythema responses were quantified as area under the curve (AUC) using Graph Pad Prism software for calculation (Figure 5A and B).

Eli spot

Ag-specifîc T cell frequency was followed with an IFN-g EL1SPOT assay (non-human primate IFN-g EL1SPOT kit; R&D Systems, Minneapolis, MN) on freshly isolated PBMC restimulated with tuberculin, according to the manufacturer’s instructions.

Briefly, capture antibody (R&D Systems, catalog number SEL961) was added in each well of a Elispot MultiScreen® HTS Filter Plates (Merck Millipore) and incubate one night at 4°C. After three washes, blocking buffer was added and plate was incubated 2 hours at ambient température. Baboons PBMC were extracted freshly from the blood of baboons by Ficoll gradient centrifugation (GE Healthcare Life Science, Paris, France). Red blood cell was then lyzed and cells washed before reconstitution at appropriate concentration in culture media (TexMacs media supplemented with penicillin-streptomycin (Gibco)) with or not tuberculin purified protein dérivative. Plate was incubated at 37°C and 5% CO2 during 18-24 hours. After three washes with wash buffer, détection antibody (R&D Systems, catalog number SEL961) was added and incubated at 4°C during 24 hours. Streptavidin-AP (R&D Systems, catalog number SEL002) was added after three washes and incubated two hours at ambient température during 2 hours. Three washes hâve been done. BCIP/NBT (R&D Systems, catalog number SEL002) hâve been added and put in the dark during 30 minutes. Several washes were necessary with wash buffer and one with deionized water (Figure 5C).

Animais

Baboons (Papio anubis ; 7-14 kg) were obtained from the Centre National de la Recherche Scientifique Centre de Primatologie (Rousset, France). The animais were housed at the large animal facility of the INSERM unit 1064. Animal studies were approved by the French National Ethics Committee.

Results in a first experiment, a first IDR with tuberculin was performed on BCG-vaccinated baboons which did not receive treatment at that time (day -30). After a washing period of one month, a second IDR was performed 4h and eveiy month after i.v. injection of 10 mg/Kg of N13B2 (white bars). A last IDR was performed after a new vaccination with BCG (14 months post- antibody injection). Control animais (black bars) received similar volume of excipient i.v. and were challenged with same protocol. Results showed that anti-lL7Ra chimeric antibody induced a very long-term protection (up to 14 months) after a single administration. Response recovered only after new vaccination.

In another experiment, a first IDR with tuberculin was performed on BCG-vaccinated baboons (dashed bars — e.g. IDR1 for V915GA). After a washing period of one month, a second IDR was performed 4h and every month after i.v. injection of 10 mg/Kg of humanized N13B2 (solid bars - e.g. IDR2-6 for

V915GA). A last IDR was performed after new vaccination with BCG and Tuberculine (dotted bars e.g. 1DR7 for V915GA). Control animais (black bars) received similar volume of excipient i.v. and were challenged with same protocol. Results showed humanized N13B2 induced also a long-term protection after a single administration in 3 out 4 evaluated baboons. “33874” animal was initially responder immédiately after antibody injection but response recovered spontaneously one month later.

Blood PBMC were re-stimulated ex vivo with tuberculin. A first IFNy elispot with or without tuberculin was performed on BCG-vaccinated baboons (dashed bars). A second IFNy elispot was performed 4 days after injection with 10 mg/Kg of humanized N13B2 (fulled bars). New ELISPOT were then performed every month at each new IDR with tuberculin in vivo. A last IFNy elispot was performed after new vaccination with BCG (dotted bars). Results showed that administration of humanized N13B2 induced antigen-specific memory T cells délétion in long-term responder animais. “33874” baboon did not showed signifîcant réduction of tuberculin-specifîc memory T cells in parallel to no long-term protection in DTH model. After new vaccination with BCG, long-term responder animais showed increased frequency of antigen-specific memory T cells which recover to basal level (before mAb injection) and this is associated with recovering of the DTH response in vivo. Altogether, these results demonstrated that long-term protection induced by humanized N13B2 is associated with antigen-specific memory T cells délétion which explaîn the long-term effect of the drug.

Example 7. Expression of IL-7, CD 127 and TSLP in IBD patients

Raw mRNA expression data obtained as detailed in Planell et al. Gut 2013 were analyzed as detailed in the legend to Figure 6.

Example 8. Signaling pathway of human PBMC stimulated with anti-CD127 antibodies plus IL7

IL7 signaling pathways were studied from lysâtes of human PBMC incubated 30 min at 37 °C with 10 pg/ml of soluble anti-human CD127 antibodies, and stimulated with IL7 (AbD Serotec, ref PHP046) at 5 ng/ml for 10 min at 37 °C. Western Blot were performed in reducing conditions with 20 pg protein of cellular lysâtes in 7.5 % polyacrylamide gels and blotted onto nitrocellulose membranes (GeHealthcare). Blots were saturated with 5 % BSA-Tris Buffer Saline (TBS) and revealed either with Phospho-Stat5, Phospho-PI3-Kinasep85, Phospho-Akt and Phospho-ERK1/2 antibody (Cell Signalling Technology) at 1/1000 in 1%BSA-TBS (ovemight at 4°C) followed by polyclonal goat anti-rabbit labeled horseradish peroxidase antibody (Cell Signalling Technology) at 1/2000 for 1 h at room température, or with GAPDH antibody (Santa Cruz) at 1/1000 in 1 % BSA-TBS (ovemight at 4 °C) followed by polyclonal goat anti-mouse labeled horseradish peroxidase antibody (Jackson Immunoresearch) at 1/2000 for 1 h at room température. Membranes were revealed by chemiluminescence using LAS-3000 imaging system (Fujifilm). The following anti-human CD 127 antibodies were thus assayed: N13B2-h3 and N13B2-hVL6 (both anti-site l/2b antibodies, disclosed herein); MD707-13-G4 (“anti-sitel antibody”, disclosed in Int. Pat. Appl. WO2013/056984) and 1A11 (disclosed in GSK patent WO2011/094259).

Example 9. Effect on T-cell cytokine release in IBD tissue

Biopsies from patients with IBD can be used as an inflammatory model of disease ex-vivo and hâve been shown here to spontaneously release high levels of proinflammatory cytokines after 24 hours of culture (UC; IFNy, 130± 19 pg/ml; IL-6, 4042 ± 529 pg/ml; IL-8, 25626 ± 1640 pg/ml - CD: IFNy, 180± 38 pg/ml; IL-6, 3653 ± 734 pg/ml; IL-8, 15540 ± 2452 pg/ml [means ±sem for UC and CD respectively]).

Anti-CD127 mAb was applied at 10 pg/ml in this organ culture assay using surgical specimens taken from inflamed colonie mucosa of 20 patients with IBD (10 with Crohn's disease and 10 with ulcerative colitis and the culture was performed at 37°C for 24 hours in medium with 1Opg/ml of IgG control mAb or blocking anti-human IL-7Ra mAb (see details of sampling and culture conditions below). A paired control specimen from each patient was sampled and cultured in the same conditions, with IgG control instead of anti-CD127 antibody. Cytokines concentration was measured by ELIS A (as detailed below) in the supematant of the ex-vivo cultured samples.

IFNy production by UC biopsy samples grown ex-vivo was significantly inhibited by anti-lL7Ra mAb. Similar results were observed for some CD biopsy samples secreting high amount of IFNy.

Ex-vivo organ sampling and culture

If mucosal resection tissue was used, small biopsy-size fragments were eut using scissors. Next, biopsies or biopsy-size fragments were placed in 300 pL serum-free HL-1 medium (Lonza, Cambridge BioScience, UK) supplemented with L-Glutamine, 100 U/mL penicillin, 100 pg/mL streptomycin and 50 pg/mL gentamycin. Mucosal expiants were incubated for 24h at 37'C and 5% CO2. The respective tested antibody (N 13B2) or IgG control used at lOpg/mL was added into the medium at the beginning of incubation time. Finally, supematant and biopsy material was snap frozen and stored at -70’C for future analysis. .

Enzyme-linked Immunosorbent (ELISA) Assay

Cytokine production in biopsy supematant was measured by enzyme-linked immunosorbent assay (ELISA). Human recombinant IFN-γ from ImmunoTools (#31673539, Friesoythe, Germany) was used accordingly to the manufacture’s instruction.

Example 10. Comparison of anti-human IL7-Ra antibodies related to their epitope characterization:

Mass spectrometry analysis: Antibody profïling using peptide microarray

The peptide Technologies' PepStarTM peptide microarrays comprise purified synthetic peptides derived from antigens or other sources that are chemoselectively and covalently immobilized on a glass surface. An optimized hydrophilic linker moiety is inserted between the glass surface and the antigen-derived peptide sequence to avoid false négatives caused by sterical hindrance. For technical reasons ail peptides contain a C-terminal glycine. Profîling experiments of samples were performed on a peptide library consisting of 52 peptides. The complété 1 ist of peptides is shown below:

SE Q ID	Sequence	SE Q ID	Sequence	SE Q ID	Sequence
44	ESGYAQNGDLEDAEL	62	F1ETKKFLL1GKSN1	80	ÎHDVAYRQEKDENKWT !
45	AQNGDLEDAELDDYS 1	63	KKFLLIGKSNICVKV	81 i	YRQEKDENKWTHVNL
46	DLEDAELDDYSFSCY ’ 1	64	iLIGKSNICVKVGEKS	\|82 ~	(KDENKWTHVNLSSTK
47	'iAELDDYSFSCYSQLË ” i	6^	SNicvkvGEKSLTck	83 ~	(kwthwlsstkltîl ^! ; 1
48	DYSFSCYSQLEVNGS	66	VKVGEKSLTCKK1DL	Î84~	\|VNLSSTKLTLLQRKL
W	SCYSQLËVNGSQHSL	67	EKSLTCKKIDLTTIV	?85~	\|sfkLTLLQRkLQPAA ’
50	QLEVNGSQHSLTCAF	68	TCKKIDLTTIVKPEA	(8k	ÎTLLQRKLQPAAMYEI i
51	NGSQHSLTCAFEDPD	69	1DLTT1VKPEAPFDL	(87	ÎRKLQPAAMYE1KVRS (
52	HSLTCAFEDPDVNTT	(70	Tl VKPEAPFDLSV1Y	(88	(PAAMYEIKVRSIPDH î
53	CAFEDPDVNTTNLEF	71	PEAPFDLSVIYREGA	*89 “ i i	(YEIKVRSIPDHYFKG i
54~	DPDVNTTNLEFEICG	72	FDLSVIYREGANDFV	'90	VRSIPDHYFkGFWSE !
55	NTTNLEFEICGALVE	73~	VIYREGANDFVVTFN	9Ï	’PDHYFKGFWSEWSPS i i
56	LEFE1CGALVEVKCL	74~	ÊGÂNDFV^FNTSHL	Î92~	(FKGFWSÊWSPSYYFR ~ ί
57	ICGALVEVKCLNFRK	— 75	DFVVTFNTSHLQKKY	93	WSEWSPSYYFRTPEI
58	LVEVKCLNFRKLQE1	76“	TFNTSHLQKKYVKVL	94	SPSYYFRTPEINNSS
59	KCLNFRKLQEIYFIE		SHLQKKYVKVLMHDV	95	YFRTPEINNSSGEMD ;

60	FRKLQEIYF1ETKKF	78	KKYVKVLMHDVAYRQ
61	QEIYFIETKKFLL1G	79	KVLMHDVAYRQEKDE

Table 5. List of peptides used in peptide microarray assays

A total of 4 samples were incubated on microarray slides using a Multiwell-format. For N13B2-h3 VL6 antibody and the other sample (MD707-13, HAL and 1A11), 6 different concentrations were applied (10 pg/ml; 2 pg/ml; 1 pg/ml; 0.1 pg/ml; 0.01 pg/ml; 0.001 pg/ml). Serial sample dilutions were incubated for 1 hour at 30°C on a multi-well microarray slide, containing 21 individual mini-arrays (1 mini-array per sample dilution). Subséquent to sample incubation, a secondary anti human IgG antibody at 1 pg/ml was added and left to react for 1 hour. An additional control incubation applying the secondaiy antibody only was performed in parallel on the same microarray slide to assess false-positive binding to the peptides. After washing and drying, the slide was scanned with a high-resolution laser scanner at 635 nm to obtain fluorescence intensity profiles. Resulting images were quantified to yield a mean pixel value for each peptide. The images were quantified to yield a mean pixel value for each peptide. Secondaiy antibody anti-h uman IgG labeled with Cy5 at 1 pg/ml.

Buffers and solutions The buffer used were TBS-buffer including 0.05% Tween20 (JPT) and Assay buffer T20 (Pierce, SuperBIock TBS T20, #37536). Acquisition and analysis were performed using Peptide microarrays (JPT Peptide Technologies GmbH, Berlin, Germany; batch #2668, Multi-Well incubation chamber, Axon Genepix Scanner 4200AL, Microarrays were scanned using a high resolution fluorescence scanner. Laser settings and applied resolution were identical for ail performed measurements. The resulting images were analyzed und quantified using spot-recognition software GenePix (Molecular Devices). For each spot, the mean signal intensity was extracted (between 0 and 65535 arbitrary units).

For further data évaluation, the so called MMC2 values were determined. The MMC2 equals the mean value of ail three instances on the microarray except when the coefficient of variation (C V) — standarddéviation divided by the mean value—is larger 0.5. In this case the mean of the two closest values (MC2) is assigned to MMC2.

Deuterium analysis

Using the HDX-2 system (Waters S.A./N.V.; Zellik, Belgium), recombinant human CD 127 and 0 or 1 molar équivalent of mAb were mixed and diluted in 99.9% D₂O, lOmM sodium phosphate, lOOmM NaCI, pH 6.8 to a final D₂O content of 90% and a CD127 or CD127/mAb complex concentration of 27.5μΜ. Hydrogen-deuterium exchange was performed at 20.0°C for 30 minutes. The exchange was quenched by a 1:1 (v/v) dilution of samples with 100mM sodium phosphate, 4M guanidineTICl, 0.4M

TCEP, pH 2.3, at 1.0 °C resulting in a final pH of 2.5. After 2 minutes, the quenched samples were loaded onto the HDX manager for online pepsin digestion at 20.0°C (Enzymate BEH Pepsin, 2.1 x 30 mm; 5μιη), followed by desalting (Acquity BEH C18 Vanguard 2.1 mm x 5 mm; 1.7 pm) and reverse phase séparation (Acquity BEH Cl8 1.0 mm x 100 mm; 1.7 μΜ) using a gradient from 5%-40% 0.2% formic acid in acetonitrile (pH 2.5) for 10 min at a flow rate of40pl/min at 0.0°C. Mass spectrometiy analysis was performed on a Waters Xevo G2-XS ESI-Q-TOF mass spectrometer in the positive ion mode, with lockspray correction. Mild source conditions (température: 90°C, capillary voltage: 2.5kV, sampling cône: 30V, desolvation gas flow: 800L/h, desolvation température: 250°C) were used in order to minimize back-exchange while ensuring proper desolvation (73). Peptide identification was assisted by collision induced dissociation collected in the MSE mode, using PLGS 3.0.2 and UNIFI 1.8. Deuterium incorporation was determined in DynamX 3.0. Structural figures were prepared using PyMOL 1.8.2.3 (Schrôdinger LLC, Cambridge, MA, USA) from PDB ID 3D13(19). Monobasic and dibasic sodium phosphate, sodium chloride, guanidine hydrochloride, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 50% sodium hydroxide, and formic acid were purchased from Sigma Aldrich (Schnelldorf, Germany) at the highest available purity. LC-MS grade solvents were sourced from Biosolve Chimie (Dieuze, France), deuterium oxide (99.9% D) and 20% deuterium chloride in deuterium oxide (99.96% D) from Cambridge Isotope Laboratories (Andover, MA, USA), hydrochloric acid 37% from VWR International (Fontenay-sous-Bois, France), and bovine cytochrome C digest from Thermo Fisher Scientifïc (Germering, Germany). Amicon ultra-centrifugal filters (0.5 mL; 10 kDa cutoff) were obtained from Merck Millipore (Molsheim, France).

Results

Epitope characterization by linear peptide array of different anti-IL7Ra mAbs identified two types of antagonist mAbs: (1) mAbs binding to the région (site-1) of interaction with IL-7, as previously described by two other groups (clone 1AI1 described in WO/2011/094259 and clone HAL described in WO/2011/104687) and including MD707-13, and (2) a mAb, i.e. N13B2-h3VL6 ofthe présent invention, which binds both site-1 and an epitope overlapping the predicted domain (site-2b) of heterodimerization between IL-7Ra and the γ-chain subunits (Walsh 2012).

N13B2-h3VL6 and MD707-13 with high and similar affinities (the binder to site-l/2b with a KD of 2.IO'^loM and a binder to site-1 with a similar KD of 5.10'¹⁰M) were recombinantly expressed with a human IgG4 Fc isotype (containing the S228P hinge mutation to prevent Fab-arm exchange) and compared to a previously described other site-1 mAb with similar afïïnity, clone 1A11, described in WO/2011/094259 (KD of 6.1O‘^,oM) and recombinantly expressed with a human IgGl Fc isotype as being developed in the clinic (NCT02293161). Analysis of conformational epitope using Hydrogen Deuterium Exchange with Mass Spectrometry (HDX-MS) conflrmed previous observations and demonstrated that the antibody of the invention, i.e. N13B2-h3VL6 (site-l/2b mAb), protected from deuterium incorporation in several peptides ofthe site-1 but also to a peptide overlapping the site-2b, while the two other mAbs (MD707-13 and 1 AI 1) signifîcantly prevent deuterium incorporation only in peptides from site-l(Data not shown).

The antibody of the invention was the only one to recognize a conformational epitope localized on site 1 from Domain 1 and a conformational epitope localized on site 2b of human CD127.

Example 11. Comparison of anti-human IL-7Ra antibodies for their ability to be agonist and/or antagonist of the IL-7 pathway.

Western Blotting

Freshly isolated human PBMCs were incubated for 30 min at 37°C with 10pg/ml of anti-human IL-7Ra mAbs, and then cultured alone or with 5ng/ml of recombinant human IL-7 (AbDSerotec) for 10min at 37°C. After stopping the reactions on ice, cells lysâtes were prepared with RIPA buffer (with Protease Inhibitor Cocktail). Proteins (15pg) were resolved under reducing conditions on 7.5% polyaciylamide gels and immobilized on nitrocellulose membranes (GeHealthcare) using standard methods. Blots were washed with 5% BSA-Tris Buffer Saline and incubated with Phospho-STAT 5, Phospho-PI3K p55 or Phospho-ERK spécifie antibodies in 1% BSA-TBS (ovemight at 4°C), followed by a polyclonal goat anti-rabbit horseradish peroxidase-labeled antibody (Cell Signalling Technology) for Ih at room température. Alternatively, blots were stained using a GAPDH antibody (Santa Cruz) in 1% BSA-TBS (ovemight at 4°C), followed by polyclonal goat anti-mouse horseradish peroxidase labeled antibody (Jackson Immunoresearch) for Ih at room température. Membranes were revealed by chemiluminescence using a LAS-3000 imaging system (Fujifilm).

RNA sequencing

Freshly isolated human PBMCs were incubated with 10pg/ml anti-human lL-7Ra mAbs (30 min at 37°C), and then cultured alone or with 5ng/ml of recombinant human IL-7 (AbDSerotec) for 3 hours at 37°C. Réactions were stopped on ice and the cell pellets resuspended inRLT buffer (Qiagen) containing 1% β mercaptoethanol in RNase/DNase free water and stored at -80°C. RNA was extracted using an RNA mini extraction kit according to manufacturer’s instructions (Qiagen). The quality and quantity of RNA were assessed by infrared spectrometry (Nanodrop) and Agilent bioanalyzer (Agilent RNA 6000 Pico Kit). Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Broad Genomics Platform according to the SmartSeq2 protocol with some modifications. Briefly, total RNA was purified using RNA-SPRI beads, polyA+ mRNA was reverse-transcribed to cDNA, and amplified cDNA was subject to transposon-based fragmentation that used dual-indexing to barcode each fragment of each converted transcript with a combination of barcodes spécifie to each sample. Sequencing was carried oui as paired-end 2x25bp with an additional 8 cycles for each index. Data was separated by barcode and aligned using Tophat version 2.0.10 with default settings. Transcripts were quantified by the Broad Technology Labs computational pipeline using Cuffquant version 2.2.1. Briefly, data were processed through CufïNorm if 50% of the reads aligned, and if at least 100,000 pairs were aligned per sample. Normalization used the default settings, including “géométrie” normalization, and expression level information as log2-transformed FPKM values (Fragments per kilobase of transcript per million mapped fragments) were used for subséquent analyses. For identification of différentiel genes, linearmodeling with estimation ofthe mean-variance relationship (limma-trend) with empirical Bayes statistical procedure were performed using the limma package in R. Genes with Benjamini and Hochberg adjusted p-value <5% and fold change (FC) >1.5 were considered as differentially expressed. For gene expression représentation, principal component analysis (PCA) and clustering were performed in R v3.3.2 using ade4/adegraphics and pheatmap packages respectively. The biological signifîcance of selected genes was assessed using the R clusterProfiler package. Gene ontology (GO) categories enriched with a false discoveiy rate (FDR) <5% and with at least fîve represented genes were selected. RNA-seq data can be accessed under GEO accession number GSE.

Results

STAT5, PIK3 and ERK signaling pathways

Anti-human IL-7Ra mAbs (N13B2-h3VL6, MD707-13,1A11 and HAL) were then compared fortheir ability to activate or block STAT5, PI3K and ERK signaling pathways previously associated with 1L7R signaling (Figures 9 and 10). As previously illustrated (see for example Figure 7), IL-7 induces potent STAT5 phosphorylation on human PBMCs and ail tested mAbs are potent inhibitors of this STAT5 phosphorylation (see Figure 7). In contrasq and as illustrated on Figures 9 and 10, the inventors found that while IL-7 induces a variable PJ3K phosphorylation and does not induce ERK phosphorylation, the antibodies of prior art (namely MD707-13, 1 Al 1 and HAL) significantly induce ERK phosphorylation and to a lesser extent PI3K signal even in the absence of exogenous IL-7 on the contrary to the antibody according to the invention. These fmdings show that anti-human lL-7Ra antibodies of prior art hâve partial agonist properties, and are therefore considered as dual agonist/antagonist mAbs for the human 1L-7R. On the contrary, an antibody according to the invention, and particularly N13B2-h3VL6, only has antagonist property for the human IL-7R.

The inventors assessed if the antibodies with agonist/antagonist properties could deliver an effective agonist signal capable of modifying human T cells. The transcriptomes of human PBMCs incubated for 3.5 hours without exogenous human IL-7, with human IL-7, and with IL-7 and an antibody (MD70713,1A11 site-1 (lgG4 #1 or IgGl #2 respectively) orN13B2-hVL6 site-l/2b (IgG4)) hâve been analyzed by RNA-based-next génération sequencing (RNA-SEQ). A total of 481 genes were differentially expressed in human PBMCs incubated with anti-human IL-7Ra mAbs compared to control conditions, while a total of334 genes were differentially expressed with human IL-7 stimulation alone compared to control conditions.

	Gene	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
logFC /Unstim	adj.P.Val	logFC /Unstim	adj.P.Val	logFC /Unstim	adj.P.Val
	ACADVL	0,85 -	- 0,040	1,15.+	0,001	K 0,87 +	0,011
5	AHR	0,86	0,029	1,16 S	0,000	1,21	0,000
	AKIRIN1	0,75	0,028	0,88	0,002	0,78	0,006
	ALDH16A1	'V 1,23 .	; 0,021	• 1,45 7	0,001	• 1,32	0,003
	ALDH5A1	0,72	0,044	0,93	0,002	1,05 ‘	0,001
	APPBP2	0,81	0,001	0,90	0,000	0,81	0,000
10	ARHGEF1	0,85	0,001	1,05 .	0,000	0,88	0,000
	B3GNT2	’ 1,03	0,034	1,23	0,003	1,15	0,005
	C17orf59	. 1,07	0,039	1,49	0,001	1,27	0,004
	CCDC117	0,77	0,024	0,88	0,002	0,80	0,005
	CCNI	0,73	0,004	0,98	0,000	0,78	0,001
15	CDK17	0,98	0,003	1,09	0,000	0,94	0,001
	COTL1	1,67 / .	; 0,000	1,80 /	0,000	1,35 ·	0,000
	CYP1B1	-0,66 - -	0,029	• · -0,75 · -	0,003	-0,69	0,006
	DDI2	1,17	0,011	1,45 ,	0,000	1,24	0,002
	DEF6	1,03	0,030	1,39	0,001	1,09	0,006
20	DNLZ	1,17	0,029	1,19	0,008	1,22	0,006
	DUSP2	. 1,73	: 0,002	'' 2,18 .	0,000	2,30	0,000
	ENG	-0,73	. 0,004	: -0,61	0,005	-0,67	0,002
	EXOC8	0,69	0,043	0,62	0,033	0,63	0,026
	FAM160B1	0,83	0,030	1,26	0,000	0,72	0,024
25	FEM1B	0,99	0,005	. 1,09	0,000	0,87	0,005
	GCFC2	1,00	0,011	0,87	0,011	0,83	0,013
	GNA15	0,99	0,011	1,14-	0,001	1,25	0,000
	GOPC	0,83	0,015	0,64	0,029	0,69	0,015
	GRSF1	0,65	0,015	0,81	0,000	0,69	0,002
30	HIPK3	0,67	0,025	0,79	0,002	0,69	0,006
	HMHAl	0,88	0,004	1,05	0,000	0,89	0,001
	HSBP1L1	1,04	0,029	0,97	0,017	0,86	0,030
	JUNB	0,91	0,025	1,25 . .	0,000	1,42 '	0,000
	KLC2	1,08	0,036	. 1,18 .	0,007	1,34 . -	0,002
35	LYSMD2	0,78	0,016	0,72	0,010	0,70	0,009
	LYZ	-0,60	0,031	-0,86	0,000	-0,76 %	0,001
	MS4A7	-0,80 Y ’	0,001	-0,98	0,000	. -0,59	0,006
	MTA2	0,84	0,038	1,18	0,001	1,10	0,002
	NCOA5	0,65	0,018	0,84	0,000	0,89	0,000
40	NSUN2	0,63	0,029	0,74	0,002	0,75	0,002
	PITHD1	0,81	0,040	. 1,03	0,002	0,81	0,013
	PLEKHF2	0,72	0,004	0,83	0,000	0,77	0,001
	POLRMT	1,09	0,001	1,30	0,000	1,06	0,000
	PPP2CA	0,75	0,025	0,87	0,002	0,75	0,007
45	PREB	0,88	0,018	0,78	0,014	0,92	0,004
	PRKCH	0,70	0,011	0,88	0,000	0,78	0,001

	PSMD3	l\!13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
1,01; ·	0,035	1,58 J	0,000	1,37 .	0,001
	PYGO2	: i,08 <.	0,003	1,07	0,001	: 0,74	0,016
	RASSFS	... 0,63 .· ;	0,004	. 0,67	0,000	.. 0,60..	0,002
5	RBL2	0,71	0,011	0,85	0,000	. 0,59	0,012
	RRP1	. 0,69 ;.	0,019	0,62	0,013	0,80	0,002
	SMCHD1	0,71	0,001	1,00 \	0,000	? 0,85.	0,000
	SREBF2	0,83	0,009	1,13	0,000	0,98	0,000
	TAF1O	1,87	0,001	1,57 .	0,001	1,28 _:	0,006
10	TAF4B	0,75	0,030	1,12	0,000	_; 1,29	0,000
	TMX4	1,04	0,009	1,13 “	0,001	0,89	0,009
	TPGS1	//.2,55/./:	0,008		0,000	2,06	0,011
	TRAM1	0,71	0,001	0,73	0,000	0,59	0,002
	TRPC4AP	0,98	0,005	1,21	0,000	. 1,06	0,001
15	TTC13	0,76	0,034	0,90	0,003	0,68	0,022
	UNC119	1,02	0,036	1,43 -	0,001	1,26	0,002
	USP9X	0,90	0,000	0,92	0,000	0,85	0,000
	VPS4A	0,74	0,042	0,95	0,002	0,75	0,012
	ZNF8O0	0,74	0,013	1,01	0,000	0,84	0,001
20	ACTN4	0,55	0,005	0,60	0,000	0,65	0,000
	C15orf48	0,63 '	0,029	-0,87	0,000	-0,51	0,035
	C3AR1	-0,56	0,011	-0,75	0,000	• -0,64	0,001
	CD247	0,80	0,004	0,87	0,000	0,50	0,038
	FAM50A	0,66	0,028	0,93	0,000	0,54	0,031
25	H CK	C -0,65	0,009	- -0,83 . ,	0,000	-0,46	0,030
	IL6ST	0,43	0,034	0,60	0,000	0,77	0,000
	JAK1	0,71 - ·	0,001	0,80	0,000	0,58	0,002
	KYNU	J -0,60 f	0,018	-0,94	0,000	-0,56	0,007
	NCF2	-0,57	0,029	-0,71 X'	0,001	-0,70 '	0,001
30	PARP10	0,58	0,037	0,75	0,002	0,73	0,002
	RAD54L2	0,61	0,037	0,66	0,008	0,53	0,033
	SMG5	0,54	0,033	0,80	0,000	ï 0,75	0,001
	TOR3A	' 0,73	0,028	0,62	0,028	0,57	0,040
	ADM	-0,88 \	0,005	-1,01	0,000	-0,02	0,967
35	CES1	-0,77 .	0,028	: -0,78	0,008	-0,45	0,145
	SLC31A2	-0,79 /	0,004	_ 0,60 /M	0,012	-0,17	0,577
	SMPDL3A	-v -1,11 .	0,009	' -1,12	0,002	-0,56	0,149
	TREM1	-0,72	0,009	-0,67	0,005	-0,28	0,287
	VNN1	-0,97	0,030	: -1,17 '	0,002	-0,35	0,423
40	FLT1	-1,01 ;	0,028	-0,66	0,101	-0,80	0,039
	ABCG1	0,39	0,339	0,70 '	0,028	0,62	0,047
	ANKRD30BL	0,88	0,342	/1,42	0,049	. . 1,73 ,	0,012
	C16orf58	0,42	0,219	0,63	0,019	0,59	0,026
	C6orfl20	0,31	0,536		0,003	0,84	0,013
45	CA2 \|	-0,33	0,450	- -0,70 .	0,033	-0,73 /	0,022

		N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
	CCNL2	0,51	0,178	- 0,75	0,012	° 0,59	0,049

	CD14	-0,64	0,070	-0,98 ·	0,001	-o,89 . :	0,002
	CDK16	0,64	0,118	- 0,96. ·	0,004	0,80	0,014
5	CHTF18	0,67	0,174	0,87 .	0,030	’ 1,13	0,004
	CLPTM1	0,74	0,111	1,06	0,005	1,01	0,007
	CREBZF	0,50	0,116	0,72	0,006	0,67	0,008
	CREG1	0,70	0,095	0,86	0,013	0,99	0,004
	CSF1R	-0,39	0,263	-0,71	0,010	-0,92 J	0,001
10	DAPK3	0,56	0,240	0,80	0,038	0,97	0,008
	DDRGK1	0,91	0,126	1,39’	0,004	_r . 1,53	0,002
	DOHH	1,40	0,061	1,45 J	0,020	? 1.69	0,006
	EMC8	0,61	0,229	1,15	0,004	1,04	0,008
	FAM115C	0,37	0,264	0,61	0,022	0,64	0,013
15	FUCA1	-0,35	0,234	. -0,59	0,011	-0,66 /	0,004
	GAPT	-0,48	0,188	/ -0,67 '	0,023	? -0,85	0,003
	GMIP	0,55	0,076	0,78	0,002	0,70	0,005
	GMPPB	0,82	0,073	0,92	0,016	0,95	0,010
	HCG11	0,70	0,087	1,05	0,002	0,80	0,014
20	HEATR3	0,43	0,280	0,74	0,019	0,74	0,017
	HEIH	0,97	0,056	1,05	0,014	0,84	0,047
	HELZ2	0,42	0,287	0,77	0,013	0,77	0,010
	HMG20B	0,54	0,088	0,61	0,019	0,59	0,022
	HNRNPAO	0,58	0,165	0,74	0,028	0,90	0,006
25	HPCAL1	0,38	0,344	0,86	0,005	0,67	0,028
	KIAA1919	0,63	0,116	0,81	0,013	0,66	0,042
	KLHDC2	0,54	0,149	0,71	0,019	0,62	0,040
	LTB	0,45	0,248	0,92	0,003	0,82	0,007
30	MAF1	0,86	0,161	. 1,54	0,002	1,38	0,005
MAFF	0,54	0,249	0,81	0,029	1,19	0,001
	MAP3K11	0,49	0,263	0,77	0,030	0,70	0,044
	MGST1	-1,05	0,089	.y. -1,24	0,015	. ' -1,06	0,036
	MIER2	0,89	0,112	0,93 : _	0,047	1,25	0,006
	MOB2	0,69	0,150	1,05	0,007	0,90	0,017
35	PET100	-0,32	0,298	. -0,61 J U	0,013	-0,61 . :	0,010
	PLK3	0,20	0,700	0,79	0,021	. 0,83	0,012
	POLG	0,52	0,086	0,97	0,000	0,79	0,002
	PSMD2	0,41	0,212	0,62	0,018	0,61	0,017
40	RAB13	-0,50	0,089	\ -0,73 .. ;	0,002	. : -0,62	0,009
RASAL3	0,74	0,067	0,92	0,006	0,72	0,028
	RELB	0,22	0,633	0,63	0,046	0,69	0,022
	RGMB	0,58	0,173	0,80	0,020	0,74	0,028
	RNF10	0,56	0,095	0,67	0,016	0,66	0,014
45	RNF149 RPL39L	0,60 -1,02	0,077 0,125	0,69 -1,09	0,016 0,049	0,69 '-ï‘4^	0,012 0,008
	RPUSD1	0,76	0,105	0,84	0,031	1,00	0,008

	SCARF1	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
0,39	0,408	0,77	0,032	. 1,13	0,002
	SENP6	0,44	0,204	0,69 .	0,012	0,72	0,007
	SIPA1L3	-0,01	0,992	.0,71	0,021	0,74 .	0,013
5	SLC25A22	0,51	0,283	' 0,75 .	0,047	0,83	0,024
	SLC38A10	0,24	0,595	- 0,76 _r	0,014	0,66 ·	0,030
	SLC43A2	0,66	0,068	0,63	0,040	0,99	0,001
	SPATC1L	0,96	0,180	LÎ7	0,044	1,22 '	0,031
	SPP1	-0,53	0,102	-0,85 . :	0,001	A -°/⁶² .	0,017
10	SRP68	0,68	0,096	0,77	0,024	0,98	0,003
	TAZ	0,95	0,092	1,23 J	0,008	1,22	0,007
	TCIRG1	0,79	0,069	: 1,15	0,001	1,15	0,002
	TNFRSF1B	0,51	0,208	0,72	0,026	0,82	0,009
	TNFRSF4	0,23	0,751	0,97	0,034	. 1,15	0,009
15	TNKS	0,66	0,052	0,78	0,006	0,67	0,016
	TSC22D2	0,47	0,137	0,65	0,011	0,61	0,014
	UBA5	0,91	0,076	1,22 ;	0,004	1,17	0,005
	USP48	0,64	0,145	0,90	0,012	0,72	0,044
	VPS51	0,50	0,145	0,82	0,003	0,76	0,005
20	ZFAND5	0,56	0,054	0,71	0,003	0,64	0,007
	ZNF259	0,47	0,242	0,85	0,007	0,65	0,038
	ZNF496	0,51	0,220	0,86	0,008	0,83	0,009
	ZNF696	0,80	0,061	1,09	0,002	0,75	0,033
	ANXA1	-0,43	0,034	-0,49	0,005	-0,63	0,000
25	C12orf75	-0,45	0,041	-0,43	0,022	-, -0,61 .	0,001
	C18orf32	-0,56	0,040	-0,61. '	0,008	-0,48	0,036
	CHMP7	0,50	0,028	0,64	0,001	0,55	0,004
	DNAJC3	-0,49	0,004	Q -0,62 /:	0,000	-0,36	0,013
	EVI2B	-0,36	0,046	-0,38	0,013	. -0,75	0,000
30	H CST	-0,54	0,034	-0,45	0,042		0,003
	HSBP1	-0,44	0,009	-0,64 · :	0,000	-0,46	0,002
	PAPOLA	0,57	0,018	0,72	0,000	0,55	0,006
	PPBP	-0,55	0,018	-0,65 ^;·	0,001	-0,50	0,010
	TIMP1	-0,52	0,019	-0,65 :	0,001	-0,57	0,002
35	TLR2	-0,42	0,044	. -0,60 .	0,001	-0,35	0,048
	UBE2D2	0,49	0,040	0,74	0,000	0,49	0,013
	AGTRAP	-0,52	0,040	. -0,66	0,002	-0,42	0,051
	CXCL16	-0,55	0,034	-0,60	0,006	-0,38	0,091
	P2RX4	-0,48	0,034	.. -0,60	0,002	-0,06	0,846
40	P2RX7	-0,72 %	0,018	-0,54	0,039	0,32	0,258
	SLAMF7	-0,44	0,011	-0,64	0,000	-0,19	0,251
	SLC2A6	-0,59	0,028	-0,56	0,013	-0,13	0,648
	SUGP2	0,64	0,005	0,50	0,012	0,33	0,114
	TNFAIP6	-0,55	0,029	-0,90 : ί	0,000	-0,13	0,637
45	TNIP3	-0,55	0,028	-0,61	0,004	-0,21	0,385

	CYBA EPG5 RNF135	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
-0,53 0,59 î -0,57	0,030 0,034 0,043	-0,39 0,39 -0,33	0,065 0,112 0,209	-0,63 , _ 0,57 _ -0,64.LJ	0,002 0,013 0,006
5	ALDH1B1	-0,79	0,036	-0,38	0,294	-0,36	0,311
	CKS2	-0,85	0,048	-0,61	0,109	-0,59	0,116
	ETFDH	-0,71	0,003	-0,36	0,090	-0,40	0,054
	FPR1	-0,87 %	0,029	-0,62	0,076	-0,29	0,467
	GSTT1	-0,94 Tp	0,030	-0,52	0,195	-0,46	0,254
10	NFYB	-0,61 A	0,025	-0,42	0,075	-0,14	0,633
	PREP	0,72	0,030	0,48	0,102	0,48	0,092
	TGFBI	-0,99 ;	0,007	-0,27	0,477	-0,36	0,310
	TMEM160	-0,75 ;	0,030	-0,30	0,373	-0,55	0,062
	TYMP	' -0,75 i	0,040	-0,38	0,278	-0,17	0,687
15	UBE3D	-0,96 T	0,046	-0,35	0,477	-0,33	0,497
	ZNF619	0,79	0,043	0,49	0,170	0,52	0,128
	ABCA1	-0,43	0,068	T. -0,60	0,002	-0,39	0,044
	ACTR6	-0,31	0,332	-0,65 T	0,007	-0,56	0,020
	ANXA5	-0,40	0,089	-0,66	0,001	-0,39	0,046
20	C16orf70	-0,21	0,548	-0,56	0,021	-0,68	0,005
	CANDI	0,54	0,102	0,56	0,046	0,88	0,001
	CAPG	-0,50	0,076	j -0,74	0,001	-0,48	0,036
	CCDC66	-0,41	0,204	-0,55	0,037	-0,70	0,006
	COMMD8	-0,33	0,165	-0,59 . ;	0,002	-0,40	0,034
25	CPD	0,52	0,055	0,46	0,043	0,64	0,004
	CYB5R1	-0,25	0,327	-0,51	0,011	-0,72 J	0,000
	DUSP6	-0,45	0,129	• -0,61	0,013	-0,48	0,049
	ENY2	-0,45	0,067	-0,46	0,026	-0,59- .	0,004
	FCER1G	-0,45	0,165	' -0,75	0,004	-0,51	0,049
30	FRMD8	0,52	0,053	0,72	0,001	0,49	0,028
	GSR	0,43	0,086	0,71	0,001	0,50	0,014
	KIF1B	0,45	0,092	0,59	0,007	0,57	0,008
	MEF2D	0,53	0,103	0,60	0,028	0,57	0,035
	MPZL1	0,38	0,220	0,58	0,020	0,75	0,002
35	MRPS6	-0,43	0,075	-0,55	0,006	' -0,88	0,000
	NDUFA5	-0,25	0,247	j . -0,60	0,000	-0,36	0,037
	NDUFB7	-0,37	0,213	-0,49	0,043	-0,62 '	0,007
	NUP153	0,34	0,228	0,57	0,009	°'⁶² . '	0,005
	S100A4	-0,41	0,120	-0,54	0,014	. -0,82 .	0,000
40	S100A8	-0,38	0,266	-0,56	0,043	-0,77	0,004
	SERPINA1	-0,46	0,067	-0,69	0,001	-0,47	0,021
	SLC17A5	-0,33	0,173	-0,51	0,010	~ -0,72	0,000
	SLC7A7	-0,47	0,076	-0,68 '	0,002	-0,52	0,016
	SNRK-AS1	0,59	0,079	0,57	0,042	. 0,59	0,031
45	5TAT5A	0,34	0,166	0,66	0,001	0,49	0,012
	TBXAS1	-0,27	0,390	-0,49	0,043	-0,64	0,007

	TFPI2	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
-0,52	0,079	' -0,65 (	0,008	-0,49	0,042
	TMEM184B	0,22	0,545	0,63	0,013	0,55	0,027

	TYROBP	-0,35	0,212	-0,56	0,012	... -0,84 .	0,000
5	USMG5	-0,39	0,120	-0,49	0,018	' -0,68	0,001
	USP19	0,48	0,145	0,76 ·	0,005	0,54	0,044
	USP38	0,44	0,105	0,49	0,029	. ^: o,6i	0,006
	XYLT2	0,27	0,329	0,60	0,005	0,57	0,006
	ACTR5	0,56	0,137	0,74	0,018	0,53	0,093
10	ADAMDEC1	-0,88	0,095	-1,24 i	0,004	-0,63	0,176
	ARHGAP18	-0,55	0,094	-0,63 -	0,021	-0,51	0,060
	AUH	1,01	0,101	1,14	0,026	0,67	0,223
	BAG2	0,29	0,548	0,70	0,042	0,42	0,254
	BAIAP2	-0,44	0,367	-0,82 ;	0,031	-0,32	0,474
15	BLOC1S3	-0,54	0,180	-0,71 ' ;	0,029	-0,37	0,290
	C16orf54	0,44	0,128	0,62	0,008	0,20	0,477
	Clorf216	0,25	0,669	0,82	0,042	0,55	0,193
	C2orf49	-0,47	0,455	-0,97 ?	0,040	-0,44	0,407
	C3orf58	0,46	0,182	0,60	0,032	0,43	0,144
20	CAMK1D	0,47	0,228	0,64	0,040	0,48	0,136
	CCL2	-0,33	0,522	-0,86	0,019	0,52	0,186
	CCL7	-0,48	0,282	-0,90	0,010	0,36	0,355
	CCRL2	-0,42	0,263	-0,76	0,011	0,09	0,848
	CD33	-1,08	0,261	-1,61	0,039	-1,28	0,103
25	CDG	0,12	0,833	0,67	0,042	0,51	0,134
	CD68	-0,35	0,184	-0,66	0,002	-0,36	0,093
	CDC42EP2	-0,30	0,490	? *0,66 .	0,037	0,08	0,864
	CLEC4E	-0,61	0,086	-0,94	0,001	-0,53	0,069
	CLEC7A	-0,35	0,521	-0,83 .	0,034	-0,51	0,223
30	CPNE8	-0,52	0,422	-1,02 λ -	0,036	-0,32	0,601
	CSF3	0,03	0,959	0,63	0,030	0,30	0,353
	CTSF	0,32	0,628	1,25	0,006	0,88	0,051
	DAB2	-0,62	0,089	-0,67	0,029	-0,49	0,119
	DFNA5	-0,63	0,080	-0,89	0,002	-0,33	0,311
35	ECU	0,62	0,167	0,78	0,031	0,45	0,249
	ELAVL1	0,56	0,120	0,74	0,013	0,51	0,091
	ERO1L	-0,52	0,086	-0,85	0,001	-0,31	0,252
	EXOSC4	-0,51	0,130	. -0,69 t ;	0,013	-0,23	0,491
	FAM105A	0,50	0,080	0,76	0,001	0,44	0,064
40	FAM83G	0,39	0,431	0,82	0,029	0,71	0,055
	FCAR	-0,32	0,280	-0,71	0,002	-0,31	0,211
	FGR	-0,41	0,067	-0,67	0,000	-0,27	0,169
	FHL3	0,57	0,180	0,72	0,035	0,24	0,572
	FKBP8	0,39	0,166	0,68	0,002	0,28	0,250
45	FLI1	0,63	0,053	. 1,03	0,000	0,51	0,065
	FPR2	-0,36	0,529	-0,88	0,032	0,11	0,867

	G0S2	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
-0,40	0,231	h -0,65 , '	0,015	0,02	0,973
	GEMIN2	-0,13	0,855	-0,85 T	0,041	-0,38	0,420
	GGH	-0,45	0,302	... -0,73 - -	0,034	-0,59	0,091
5	GIMAP1	0,39	0,256	0,69	0,012	0,47	0,097
	GIMAP1-
	GIMAP5	1,89	0,075	1,77 .	0,047	0,95	0,331
	GNG1O	-0,30	0,280	-0,61.	0,005	-0,10	0,752
	GPR137B	-0,51	0,376	-0,90	0,044	-0,31	0,566
10	GPR35	-0,41	0,322	-0,70' _:	0,031	-0,12	0,791
	GPR84	-0,38	0,154	-0,68 ‘	0,002	0,05	0,891
	GTF2I	0,85	0,101	. 1,11	0,009	0,75	0,082
	GZMM	0,55	0,215	0,77	0,029	0,31	0,448
	H B EGF	-0,57	0,080	-0,76	0,004	0,02	0,970
15	HECTD3	0,61	0,107	0,66	0,038	0,38	0,267
	HLCS	0,46	0,310	0,80	0,024	0,59	0,105
	IFF01	0,50	0,145	0,61	0,032	0,23	0,487
	IGSF6	-0,65	0,058	A -0,85	0,003	-0,23	0,502
	IL19	-0,62	0,543	-1,55	0,033	-0,56	0,516
20	IL1R2	-0,38	0,627	. -1,20	0,025	-0,63	0,285
	IL24	-0,86	0,053	$ -0,92	0,013	-0,51	0,201
	IL6	-0,43	0,196	-0,68 .	0,012	0,14	0,701
	ILF3-AS1	-0,50	0,263	-0,76	0,035	-0,26	0,542
	ISY1-RAB43	0,18	0,841	-1,07	0,033	0,24	0,717
25	JTB	0,24	0,621	0,73	0,030	0,33	0,372
	KCNA3	0,08	0,879	0,85	0,005	0,37	0,264
	KMO	-0,50	0,122	-0,72	0,006	-0,38	0,183
	LACC1	-0,40	0,071	\ -0,65	0,000	-0,27	0,171
	LILRB3	-0,31	0,410	-0,67	0,017	0,12	0,763
30	MET	-0,36	0,419	-0,67	0,049	-0,06	0,916
	MMPI	-0,29	0,342	-0,74	0,001	-0,29	0,245
	MMP19	-0,34	0,254	: -0,59 .	0,013	-0,32	0,204
	MTERFD1	-0,02	0,982	,-0,61	0,048	-0,38	0,249
	N CFI	-0,48	0,214	-0,85.	0,006	-0,39	0,241
35	NCF1C	-0,56	0,254	-1,07	0,005	-0,53	0,198
	NENF	0,90	0,068	0,97	0,018	0,51	0,247
	0CEL1	0,70	0,198	1,04	0,018	0,57	0,232
	PEX26	0,54	0,179	0,83	0,010	0,47	0,166
	PGAM5	0,47	0,256	0,82	0,013	0,63	0,055
40	PHF1	0,08	0,883	0,65	0,043	0,38	0,270
	PIGP	-0,03	0,970	' -0,68 ’	0,046	-0,24	0,557
	PILRA	-0,49	0,095	- -0,75	0,002	-0,45	0,063
	PKIA	0,57	0,256	0,86	0,034	0,54	0,211
	PLA2G7	-0,53	0,058	-0,73	0,002	-0,33	0,187
45	PLAUR	-0,32	0,229	-0,68 /	0,001	-0,21	0,363
	PMP22	-0,72	0,132	. -1,08	0,005	-0,45	0,290

	PTAFR	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
-0,58	0,099	-0,73	0,012	0,08	0,867
	QPCT	-0,57	0,108	\ -0,81 Λ	0,005	-0,53	0,072
	RAB2A	0,45	0,198	0,60	0,033	0,53	0,059
5	RAC1	0,90	0,065	1,13	0,005	0,76	0,063
	RALY	0,39	0,234	0,60	0,023	0,44	0,107
	RANBP10	0,14	0,738	0,59	0,026	0,31	0,293
	RASA3	0,38	0,120	0,66	0,001	0,34	0,102
	RAVER1	0,43	0,182	0,62	0,017	0,50	0,053
10	RCN2	0,48	0,231	0,76	0,017	0,41	0,234
	RETSAT	0,48	0,231	0,65	0,047	0,59	0,065
	RSPH3	-0,13	0,841	• -0,95 ;	0,008	-0,29	0,497
	SEMA4A	-0,53	0,057	’ -0,69 )	0,003	-0,04	0,911
	SEPHS2	0,43	0,503	1,00	0,030	0,87	0,055
15	SLAMF8	-0,27	0,544	-0,64	0,040	0,53	0,087
	SNAPC1	-0,34	0,325		0,016	-0,30	0,300
	SUSD3	0,53	0,101	0,59	0,027	0,41	0,136
	TAFIA	-0,23	0,588	: -0,60	0,043	-0,03	0,954
	TBC1D17	0,37	0,330	0,65	0,029	0,38	0,227
20	TBC1D22A	0,38	0,254	0,62	0,020	0,23	0,469
	TBC1D25	0,37	0,298	0,71	0,010	0,44	0,120
	TBL2	0,31	0,450	0,68	0,026	0,23	0,542
	TFE3	0,23	0,552	0,61	0,027	0,41	0,159
	THBS1	-0,37	0,256	-0,64	0,012	-0,49	0,053
25	TLR8	-0,74	0,089	-0,88 ;	0,015	-0,03	0,963
	TMEM176B	-0,94	0,068	-1,03 :	0,016	-0,82	0,055
	TPRKB	-0,46	0,212	\ -0,88	0,003	-0,48	0,120
	UBE3A	0,06	0,924	0,65	0,042	0,54	0,096
	USP21	0,37	0,272	0,75	0,004	0,27	0,363
30	VAMP4	-0,31	0,375	-0,61	0,024	-0,40	0,163
	VNN2	-0,47	0,086	ΐ -0,60	0,008	-0,41	0,074
	WBP5	-0,97	0,205	^1,42 “ .	0,020	-0,70	0,298
	XPOT	0,78	0,061	0,78	0,026	0,57	0,108
	ZDHHC3	0,56	0,095	0,73	0,008	0,30	0,328
35	ZER1	0,58	0,161	0,78	0,019	0,36	0,333
	ZNF589	0,27	0,504	0,73	0,012	0,25	0,474
	ZNF71	0,55	0,101	0,65	0,020	0,23	0,487
	ZNF792	0,70	0,108	0,95	0,008	0,67	0,063
	ABHD14A	-0,24	0,527	0,00	1,000	-0,62	0,018
40	ACSL1	0,07	0,872	-0,08	0,823	0,62	0,014
	AGO2	0,21	0,547	0,31	0,259	0,71	0,003
	APOBEC3D	0,20	0,673	0,12	0,801	0,68	0,034
	ARHGAP31	0,22	0,582	0,28	0,364	0,60	0,026
	ATF3	0,18	0,771	0,04	0,949	/ 0,89	0,017
45	B3GNT5	0,11	0,855	-0,01	0,985	0,81	0,013
	BACH2	0,26	0,352	0,25	0,296	0,71	0,001

		N13B2-h3VL6	MD707-13 lgG4#l	1A11 IgGl #2
	C19orf43	-0,57	0,055	-0,30	0,285	y -0,59	0,014
	C9orf69	0,34	0,319	0,40	0,153	0,64	0,012
	CCL4	-0,15	0,757	-0,39	0,248	.0,94.	0,002
	CCL8	-0,28	0,813	-0,94	0,214	7 2,02 / /	0,003
5	CCR2	-0,56	0,174	-0,32	0,412	- -1,00 :	0,002
	CD 160	-0,64	0,288	-0,74	0,143	. 2,02 , ;	0,000
	CD274	-0,28	0,494	-0,36	0,285	0,78	0,008
	CD36	-0,77	0,172	-0,72	0,129	-1,26 ,	0,005
	CD52	-0,08	0,769	-0,25	0,155	-0,61	0,000
10	CD69	0,02	0,974	-0,08	0,770	0,82	0,000
	CD72	-0,30	0,499	-0,29	0,431	0,65	0,038
	CH K A	-0,41	0,410	-0,56	0,156	-0,76 .	0,039
	CLIC3 COMMD3-	0,05	0,939	-0,05	0,913	-0,67	0,041
15	BMI1	0,34	0,655	0,21	0,771	-1,41 ?	0,005
	COX17	-0,21	0,616	-0,45	0,133	...... -0,67 .	0,015
	CRTAM	-0,39	0,388	-0,39	0,310	1,05	0,002
	CSF2	0,11	0,939	0,79	0,337	1,61 “	0,024
	CSF2RB	0,04	0,930	0,01	0,989	0,73	0,004
20	CX3CR1	-0,19	0,662	-0,24	0,493	. -0,64	0,023
	CXCL10	-0,55	0,511	-0,36	0,648	. 1,58	0,007
	CXCL9	-0,32	0,687	-0,05	0,956	1,61	0,002
	DUSP1	0,85	0,133	0,82	0,088	0,93	0,043
	DVL2	0,54	0,143	0,58	0,058	0,61	0,040
25	EGR2	-0,67	0,094	-0,45	0,210	0,85	0,008
	EIF4EBP1	-0,46	0,260	-0,15	0,737	-0,81 .	0,010
	EPSTI1	0,09	0,874	0,16	0,728	0,75	0,021
	FAM91A1	0,35	0,338	0,50	0,083	0,59	0,034
	FASLG	0,05	0,933	0,06	0,881	0,76	0,007
30	FBXO30	0,27	0,586	0,63	0,077	0,76	0,024
	FGFBP2	-0,16	0,647	-0,23	0,384	-0,77	0,001
	FUS	0,30	0,429	0,37	0,238	0,61	0,029
	GBP1	-0,22	0,716	-0,34	0,475	1,07	0,005
	GBP1P1	-0,19	0,820	-0,02	0,979	1,11	0,019
35	GBP2	0,04	0,924	0,02	0,956	0,61	0,004
	GBP4	0,00	0,997	0,08	0,823	0,74	0,002
	GBP5	0,00	1,000	0,06	0,921	1,14	0,002
	GCHFR	-0,03	0,952	-0,16	0,574	-0,64 . .	0,004
	GIMAP8	0,37	0,298	0,49	0,092	0,59	0,035
40	GZMA	-0,28	0,298	-0,29	0,194	, -0,61 .	0,003
	GZMB	0,05	0,916	-0,08	0,829	0,71	0,002
	GZMH	-0,23	0,512	-0,36	0,181	-0,68 ;	0,005
	HAVCR2	0,07	0,858	-0,19	0,435	0,66	0,001
	HIST1H2BK	-0,29	0,391	-0,47	0,076	-0,71	0,005
45	HIVEP1	0,36	0,213	0,09	0,783	0,65	0,005

	HSPBPl	N13B2-h3VL6	MD707-13 lgG4 #1	1A11 IgGl #2
0,53	0,153	0,49	0,120	0,78 y	0,007
	HSPH1	0,40	0,414	0,71	0,056	0,72	0,046
	IFNG	0,32	0,706	-0,32	0,654	.- 1,21 ^;	0,024
5	IGF2R	0,15	0,664	0,37	0,119	. 0,66	0,003
	IKZF2	0,20	0,664	0,42	0,212	L 0,61- .	0,049
	IL21R-AS1	0,16	0,635	0,34	0,172	. .. 0,61	0,007
	INADL	0,56	0,070	0,45	0,088	. 0,59	0,018
	INHBA	-0,08	0,885	-0,24	0,549	0,89	0,005
10	INIP	-0,50	0,067	-0,38	0,111	./ -0,59 ·	0,008
	INTS3	0,55	0,092	0,49	0,079	0,67	0,010
	IRF1	0,01	0,996	0,13	0,727	0,68	0,013
	IRF4	0,36	0,256	0,41	0,113	1,03	0,000
	IRF8	-0,09	0,790	0,01	0,982	0,71	0,000
15	IRG1	-0,36	0,567	-0,45	0,380	1,41	0,002
	ISOC2	0,30	0,564	0,07	0,898	0,74	0,036
	JAK2	0,63	0,087	0,53	0,091	0,98	0,001
	JUN	0,06	0,880	0,23	0,400	0,76	0,002
	KCNK6	0,37	0,503	0,60	0,144	0,80	0,040
20	KDM6B	-0,05	0,911	0,19	0,527	0,66	0,006
	KHK	-0,81	0,070	-0,17	0,733	-0,73	0,047
	KIR2DL4	-0,26	0,656	-0,39	0,402	1,02	0,008
	KLRB1	-0,13	0,622	-0,26	0,178	-0,82	0,000
	LGALS1	-0,42	0,178	-0,49	0,052	-0,69	0,005
25	LIMK2	-0,08	0,879	-0,01	0,976	0,79	0,006
	LMNB1	0,13	0,793	0,30	0,418	0,72	0,018
	LOC729013	-0,42	0,503	-0,39	0,469	ÿ -0,93 .	0,035
	LRCH4	0,72	0,170	0,74	0,087	0,89	0,033
	LST1	-0,45	0,432	-0,83	0,055	-1,03	0,013
30	MAP3K13	-0,09	0,837	-0,38	0,125	-0,64	0,006
	MB21D1	0,00	1,000	0,15	0,701	0,76	0,009
	MFGE8	0,08	0,941	0,21	0,792	'-1,17	0,034
	MPPE1	-0,31	0,477	-0,43	0,220	-0,77 . _:	0,013
	NAB2	0,25	0,731	0,55	0,278	1,23	0,006
35	NDUFAF6	-0,27	0,557	-0,54	0,115	-0,65 :< :	0,044
	NFKBIB	0,08	0,881	0,21	0,576	0,60	0,043
	NOP14	0,14	0,821	0,55	0,164	0,80	0,028
	NR4A1	0,12	0,777	0,06	0,871	0,95	0,000
	NR4A2	-0,09	0,841	-0,25	0,373	0,75	0,002
40	OSBPL5	-0,55	0,152	-0,21	0,581	-0/62	0,046
	PAGR1	0,48	0,159	0,54	0,051	0,64	0,016
	PIM3	0,05	0,930	0,42	0,181	0,59	0,040
	PLEK	-0,08	0,853	-0,17	0,579	0,82	0,001
	PML	0,51	0,168	0,56	0,067	0,62	0,036
45	PPP1R15B	0,27	0,302	0,32	0,143	0,60 :	0,003
	PTGES2	-0,39	0,545	-0,64	0,189	-1,00 .	0,026

	N13B2-	h3VL6	MD707-1	3lgG4#l	1A11 Ig	;G1#2
PTGS2	0,12	0,854	-0,02	0,971	0,79	0,022
PYCARD	-0,16	0,703	-0,21	0,550	” < -0,60	: 0,032
RAB20	-0,28	0,509	-0,34	0,324	0,61	0,045
RGS16	-0,16	0,809	-0,07	0,901	1,09	0,004
RNF19A	0,22	0,457	0,26	0,282	0,67	0,002
RPL21	-0,54	0,132	-0,10	0,809	. -0,84 ?	.i 0,004
RPL34	-0,07	0,837	-0,10	0,709	-0,59	- 0,003
RPS29	-0,02	0,975	-0,06	0,878	-0,60 '	U 0,024
SBNO2	0,14	0,758	0,23	0,515	0,59	0,044
SERPINE2	-0,19	0,655	0,02	0,957	0,63	0,026
SH2D1B	-0,11	0,749	-0,09	0,742	0,94	0,000
SH3BGRL3	-0,28	0,207	-0,26	0,180	' -0,66 T	J 0,000
SLC25A20	-0,14	0,731	-0,33	0,257	-0,59	i 0,024
SLC7A5	0,33	0,355	0,21	0,530	0,60	_ °'⁰²⁴
SLMO2-ATP5E	-0,30	0,893	-0,82	0,594	:.-2,45	I 0,042
SMG6	0,30	0,410	0,45	0,121	0,62	0,021
SM1M11	-0,40	0,417	-0,43	0,299	: -0,74	0,045
SMIM14	-0,32	0,165	-0,36	0,057	-0,68	0,000
SNHG8	-0,25	0,666	-0,37	0,400	-0,80	0,034
SPRY2	-0,16	0,880	-0,46	0,531	1,37	0,018
SRC	0,25	0,549	0,32	0,341	0,59	0,042
SREBF1	0,41	0,509	0,58	0,233	0,93	0,034
STARD4	0,21	0,583	0,33	0,268	0,83	0,002
STAT1	0,20	0,582	0,23	0,428	0,64	0,008
SUMF1	-0,17	0,683	-0,37	0,222	-0,64 -	0,017
TAGAP	0,03	0,937	0,08	0,793	1,06	0,000
TESK1	-0,06	0,921	0,18	0,645	0,63	0,038
TMEM165	0,42	0,303	0,57	0,083	0,65	0,038
TNF	-0,33	0,261	-0,32	0,206	0,61	0,007
TNFRSF10A	0,34	0,619	0,63	0,215	0,93	0,044
TNFRSF9	0,02	0,976	-0,09	0,807	1,08	0,000
TP53I13	0,53	0,210	0,62	0,073	0,84	0,010
TYSND1	-0,24	0,590	-0,10	0,818	-0,70	0,018
UBE2D1	0,63	0,111	0,62	0,066	0,91	0,005
XCL1	-0,07	0,941	0,02	0,979	2,19	) 0,000
XCL2	-0,25	0,805	0,77	0,257	/2/83)	3 0,000
ZNF326	-0,37	0,344	-0,53	0,088	-0,60 U	: 0,044

Table 6. List of genes significantly (FDR 5%) and differentially (fold-change >1.5) expressed after incubation of human PBMCs (n=7) with anti-IL7Ra mAbs compared to unstimulated cells.

Venn diagram (Figure 1 IC) analysis identifîed 61 common difTerentially expressed genes with the three mAbs without any particular GoMiner gene ontology enrichment. Despite 61 common genes, the antibody of the invention induces only 31 significant gene modifications without gene ontology enrichment. In contrast, the two antibodies of prior art induce an important transcriptional modification of human PBMCs transcriptome, with 245 difTerentially expressed genes induced by the site-1 IgG4 #1 mAb and 237 difTerentially expressed genes induced by the site-1 IgGl #2 mAb. 78 difTerentially expressed genes were common between these two site-1 mAbs, but these genes are not difTerentially expressed when the PBMCs are stimulated with N13B2-hVL6.

Principal Component Analysis (PCA) of the IL-7 signature shows that the difTerentially expressed genes induced by the antibodies of the prior art are strongly different from the difTerentially expressed genes induced by IL-7. GoMiner gene ontology enrichment suggests that both antibodies of the prior art modifÿ biological PBMCs functions such as leukocyte activation, prolifération, migration, chemotaxis, cytokine sécrétion and inflammatoiy responses associated with the MAPK/ERK pathway.

Among the 334 genes difTerentially expressed with 1L7 compared to the control condition, 93 genes were difTerentially expressed with a high fold-change (>2) and were separated into 3 distinct clusters by heat-map analysis (Figure 11A). The first cluster of downregulated genes assessed by gene ontology enrichment were mainly implicated in leukocyte différentiation and apoptosis, a second cluster of highly upregulated genes were associated with leukocyte adhesion, différentiation and activation, and a third cluster of upregulated gene had mixed gene ontology. Interestingly, ali tested mAbs (both site-1 or sitel/2b) exhibited similar effects by preventing cluster-1 and cluster-2 modification but without impact on cluster-3 (Figure 11 B).

Altogether, transcriptional analyses confirmed that despite site-1 and site-1/2b anti-human IL-7Ra mAbs shared similar antagonist properties, the two site-1 mAbs described in the State of the art induced significant transcriptional modifications of human PBMCs compatible with T-cell activation and inflammatoiy responses induced by the MAPK/ERK. pathway.

The site-l/2b anti-human IL-7Ra mAb of the invention, i.e. N13B2-hVL6, induced less transcriptional modification of human PBMCs compared to the two site-1 mAbs described in the State of the art.

The lack of spécifie and “antagonist-only” anti-IL-7Ra mAbs for laiger species has prevented vérification of this effect in primates or humans. The inventors found that the agonist/antagonist properties of anti-IL-7Ra mAbs dépends on the spécifie epitope targeted, since antibodies of the State of the art binding the site-1 IL-7 interaction domain appear to hâve both agonist/antagonist properties whereas the antibody of the invention binding the dimerization domain of lL-7Ra/yc (site 2b) display strict antagonist activity. The inventors suggest that the antibody of the invention could perturb IL7Ra/yc dimerization required for receptor intemalization and signaling. “Antagonist-only” antibodies against the 1L-7R prevented long-term memory T-cell-mediated skin inflammation in primates, even after chronic antigen stimulation, without inducing lymphopenia or polyclonal T-cell functional or metabolic deficiencies.

1L-7 has been shown to induce proliférative and anti-apoptotic signais through 1L-7R signaling mainly by activating the JAK/STAT pathway. IL-7R signaling is also believed to involve the PI3K/AKT pathway, but this has been observed in transformed immortalized cell Unes or primary thymocytes and these signais were not détectable in peripheral naïve or memory human T lymphocytes (Watanabe, J. Exp. Med, 1998). Several reports hâve also suggested that IL-7R signaling could amplify ERK phosphorylation either in T lymphocytes or pro-B cell subsets (Deshpande P. 1. Immunol, 2013; Fleming HE and Paige CJ, Immunity, 2001). The rôle of these pathways in IL-7R signaling in mature and human T cells is less clear. Using primary freshly isolated human PBMCs (mostly composed of T and B lymphocytes, monocytes and NK cells) from healthy volunteers stimulated with a high concentration of recombinant human 1L-7 (5000pg/ml, while the concentration in sera is — 5 pg/ml in non-lymphopenic conditions Wong H-L, Cancer Epidemiol Biomarkers Prev, 2008)), the inventors confïrmed that IL-7 induced reproducible STAT5 phosphorylation. PI3K signal activation was more variable, and they did not observe any ERK phosphorylation. While ail anti-IL7Ra mAbs used in this study were potent inhibitors of lL-7-inducedpSTAT5 and displayed similartranscriptional antagonist properties, we found that two site-1 mAbs of the state of the art induced PI3K/ERK agonist signais and important transcriptîonal modifications associated with T-cell activation and inflammatory responses induced by the MAPKZERK pathway. These opposing dual agonist/antagonist properties of some mAbs are not unique since other targets such as EL-4, EL-6R, IL-15, CD28, CD38, CD40, or HER2 demonstrated similar activities after receptor endocytosis/intemalization.

The heterodimerization of this site 2b with TSLPR has also been recently confïrmed and demonstrated to be poised for receptor signaling as already predicted for lL-7Ra and the γ-chain (Verstraete K„ Nature Communications, 2017). Interestingly, the predicted heterodimerization site between EL-7Ra/y-chain and IL-7Ro/TSLPR is overlapping suggesting a shared heterodimerization-mediated signaling mechanism between both receptors.

We found that the in vitro pSTAT5 inhibition assay was not prédictive of anti-IL-7R antibody efficacy in vivo. In a previous report, another anti-IL7Ra mAb prevented in vitro and ex vivo IL-7-induced pSTAT5 in primates but did not protect from brain inflammation in an experimental autoimmune encephalitis (EAE) marmoset model (Dunham J., J. Neuroimmune.Pharmacol, 2016).

IL-7 has been well-described in maintaining the pool of peripheral naïve and memory T lymphocytes in mice. In primates however, the importance of EL-7 in maintaining peripheral T-cell homeostasis might therefore be less évident and/or redundant mechanisms might explain the différence between species.

These data showed that antagonist properties of anti-IL-7Ra mAbs and in-vivo efficacy are not only related to the prévention of IL-7 binding and pSTAT5 inhibition. These data showed that the antibody of the invention also targeting the receptor heterodimerization site (site 2b), is “antagonist-only” and resuit in a higher efïicacy of inhibitory T cell responses in vivo.

Targeting 1L7R with “antagonist-only” antibodies has the potential to regulate antigen-specific memoiy T cell survival and accumulation, and therefore might promote the prévention of long-term relapse in 5 autoimmune and inflammatoiy diseases.

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Claims

Claims

1. An antibody or an antigen-binding fragment thereof, which specifically binds to CD127, in particular to human CD 127, comprising:

- an antibody light chain comprising or an antibody light chain variable domain consisting of a sequence selected from the group consisting of SEQ ID No: 9; SEQ ID No: 10; SEQ ID No: 11; SEQ ID No: 12; in particular SEQ ID No: 12; and

- an antibody heavy chain variable domain consisting of the sequence set forth in SEQIDNo: 7.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein said antibody is a humanized monoclonal antibody, in particular wherein the antibody light chain constant domain is derived from a human kappa light chain constant domain, in particular wherein the light chain constant domain consists of the sequence of SEQ ID No:27 or 28, and wherein the antibody heavy chain constant domain is derived from a human IgGl, IgG2, IgG3, or IgG4 heavy chain constant domain, in particular from a human IgG4 heavy chain constant domain, in particular wherein the antibody heavy chain constant domain consists of the sequence with SEQ ID No:26.
3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody light chain comprises or the antibody light chain variable domain consists of SEQIDNo: 12.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is an antagonist of IL-7R signalling induced by IL-7 and which does not induce the activation of the phosphatidylinositol 3-kinase and/or of the ERK signalling pathway.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which recognizes an epitope comprising a sequence taken from the 2b site of CD 127 and/or disrupts the binding of CD127 to the gc common chain of cytokine receptors, in particular which recognized at least the third beta sheet of the site 2b of CD 127.
6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which does not induce the intemalization of CD 127 and/or inhibits IL7-induced intemalization of CD127.
7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which does not increase the maturation of dendritic cells induced by TSLP.
8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which has a long-lasting effect and/or a fast effect, in particular which has a fast local effect.
9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which specifically binds to human CD 127 with an affinity constant KD lower than 5E-9 M, as may be determined by biosensor analysis.
10. A combination of isolated nucleic acid molécules encoding an antibody or antigenbinding fragment thereof according to any one of claims 1 to 9, in particular a first isolated nucleic acid molécule comprising or consisting of a sequence selected from the group consisting ofSEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17 and SEQ ID No: 18; and a second isolated nucleic acid molécule comprising or consisting of the sequence of SEQ ID No: 13.
11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and/or the combination of isolated nucleic acid molécules according to claim 10, and a pharmaceutical vehicle.
12. The pharmaceutical composition according to claim 11, which is suitable for local administration, in particular for local subcutaneous, enteric or oral administration, more particularly for delivery to the colon.
13. A kit comprising a pharmaceutical composition of claim 11 or claim 12 and a delivery device suitable for local administration, in particular a subcutaneous, enteric or oral delivery device, more particularly said device comprising a pre-filled syringe or a needle-free device.
14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the combination of isolated nucleic acid molécules according to claim 10, or the pharmaceutical composition according to claim 11 or 12, or the kit of claim 13, for use as a médicament.
15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to

9, or the combination of isolated nucleic acid molécules according to claim 10, or the pharmaceutical composition according to claim 11 or 12, or the kit of claim 13, for use according to claim 14 in the prévention or treatment of organ or tissue transplant rejection or of a disease selected from the group consisting of autoimmune diseases, particularly rheumatoid arthritis, systemic sclerosis, multiple sclerosis, type I diabètes, 5 autoimmune thyroiditis, systemic lupus erythematosus, primary sjôgren syndrome, and inflammatory diseases, particularly inflammatory bowel disease (IBD), more particularly Crohn’s disease and ulcerative colitis and encephalomyelitis and allergie diseases and cancer diseases and diseases related to transplantation and respiratory diseases.