Anti-Mullerian Inhibitory Substance Antibodies and Uses Thereof

Abstract

In ovarian malignancies, the Müllerian inhibitory substance (MIS) type II receptor (MISRII) and the MIS / MISRII signaling pathway are promising therapeutic targets. Conversely, the role of three MIS type I receptors (MISRIs; ALK2, ALK3, and ALK6) in this cancer remains to be elucidated. Using four ovarian cancer cell lines and ovarian cancer cells isolated from patient tumor ascites, we found that ALK2 and ALK3 are the two major MISRIs involved in MIS signaling at low and high MIS concentrations, respectively. Furthermore, high MIS concentrations were associated with apoptosis and reduced clonogenic survival, whereas low MIS concentrations improved cancer cell survival. Finally, we demonstrated that the anti-MIS antibody B10 inhibited the MIS-promoting effect. These final results pave the way for innovative therapeutic approaches that suppress the MIS-proliferative effect, instead of administering high doses of MIS to induce cancer cell apoptosis.

Description

[Technical field]

[0001] Field of the invention: The present invention relates to anti-Mullerian inhibitory substance (MIS) antibodies and their use in the treatment of MIS- or MISRII-positive cancers, in particular gynecological cancers, lung cancer or colorectal cancer, in a subject in need thereof.

[0002] Background of the invention: Müllerian inhibitory substance (MIS) is a member of the TGFβ family that acts by binding to its specific receptor (MIS type II receptor; MISRII) to recruit type I receptors (MISRI: ALK2, ALK3 and ALK6). MISRI phosphorylation induces SMAD1 / 5 / 8 phosphorylation and their nuclear translocation, which, together with SMAD4, regulate different responsive genes depending on the target tissue (di Clemente et al., 2010; Josso and Clemente, 2003). In vitro and in vivo preclinical findings as well as data from clinical samples (Bakkum-Gamez et al., 2008; Masiakos et al., 1999; Meirelles et al., 2012; Pepin et al., 2015; Renaud et al., 2005; Wei et al., 2010) demonstrated that MISRII and the MIS / MISRII signaling pathway are promising therapeutic targets in gynecological tumors, particularly ovarian malignancies (reviewed in (Kim et al., 2014)). This signaling cascade could be targeted using recombinant MIS or anti-MISRII antibodies. However, the use of recombinant MIS has been hampered by issues regarding the production and delivery of sufficient amounts of bioactive MIS to tumor sites (Donahoe et al., 2003). Recently, Pepin et al. described an alternative delivery approach (not yet in clinical phase) using a unique production strategy and gene therapy (Pepin et al., 2013, 2015). Among anti-MISRII antibodies (Salhi et al., 2004) and antibody fragments (Yuan et al., 2006, 2008), the monoclonal antibody (MAb) 12G4 and its humanized version have been extensively evaluated in preclinical studies (Bougherara et al., 2017; Estupina et al., 2017; Gill et al., 2017; Kersual et al., 2014), and a humanized antibody (GM-102 or murlentamab) is currently being tested in clinical trials (NCT02978755, NCT03799731).The mechanism of action of glycoengineered murlentamab involves antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell phagocytosis, but little apoptosis, suggesting that its action is not directly related to the MIS signaling pathway (Bougherara et al., 2017; Estupina et al., 2017). Indeed, in MISRII-positive cancer cells, MIS inhibits proliferation and induces apoptosis.

[0003] To understand why MIS signaling pathway is not involved in the mechanism of action of this anti-MISRII MAb, we analyzed the role of three MISRIs (ALK2, ALK3 and ALK6) in ovarian malignant tumor cell lines and malignant tumor cells isolated from ascites samples of ovarian malignant tumor patients.Indeed, the role of ALK2, ALK3 and ALK6 in several types of cells has been studied in developmental stages and other physiological conditions (Belville et al., 2005; Clarke et al., 2001; Josso et al., 1998; Orvis et al., 2008; Sedes et al., 2013; Visser et al., 2001; Zhan et al., 2006), but there is little data available in cancer. Basal et al. demonstrated that MISRII, ALK2, ALK3 and ALK6 are expressed in epithelial ovarian cancer (immunohistochemical analysis of 262 samples) but could not assess their specific role ( Basal et al., 2016 ).

[0004] Herein, we found that ALK2 and ALK3 are the two major MISRIs used in MIS signaling in four ovarian cancer cell lines (two from epithelial ovarian tumors and two from sex cord-stromal tumors, including one from granulosa cell tumor), and that they have differential roles according to MIS concentration. We then showed that the promotion of cancer cell survival by low concentrations (0.5-13 nM or less) of MIS can be inhibited using MIS siRNA and a new anti-MIS antibody, B10. This observation paves the way for innovative therapeutic approaches to suppress the MIS proliferative effect, instead of administering high doses of MIS to induce apoptosis.

[0005] Summary of the invention: In ovarian malignancies, Müllerian inhibitory substance (MIS) type II receptor (MISRII) and the MIS / MISRII signaling pathway are promising therapeutic targets. Using four ovarian cancer cell lines and ovarian cancer cells isolated from patients' tumor ascites, we found that ALK2 and ALK3 are the two major MISRIs involved in MIS signaling at low and high MIS concentrations, respectively. Furthermore, high MIS concentrations were associated with apoptosis and reduced clonogenic survival, whereas low MIS concentrations improved cancer cell survival. Finally, we showed that MIS siRNA and anti-MIS antibody B10 inhibited the MIS pro-survival effect. These last results pave the way for innovative therapeutic approaches to suppress MIS proliferative effects, instead of administering high doses of MIS to induce cancer cell apoptosis.

[0006] Accordingly, the present invention relates to anti-Mullerian inhibitory substance (MIS) antibodies and their use in the treatment of MIS or MISRII positive cancers in subjects in need thereof. In particular, the present invention is defined by its claims.

[0007] Detailed description of the invention: definition As used herein, the term "Mullerian inhibitory substance" or "MIS", also known as "anti-Mullerian hormone" or "AMH", has its general meaning in the art and refers to a glycoprotein hormone structurally related to inhibin and activin from the transforming growth factor beta (TGFβ) superfamily, which plays an important role in growth differentiation and follicle formation. MIS is a 140 kDa dimeric glycoprotein encoded by the AMH gene on human chromosome 19p13.3. Its entrez reference number is 268 and its Uniprot reference number is P03971. MIS acts by binding to its specific MIS type II receptor (MISRII or AMHR2) to recruit the type I receptor (MISRI or AMHR1).

[0008] As used herein, the term "Mullerian inhibitory substance type II receptor" or "MISRII", also known as "AMHR2", has its general meaning in the art. MISRII is encoded by the AMHR2 gene on human chromosome 12q13.13. Its entrez reference number is 269 and its Uniprot reference number is Q16671.

[0009] As used herein, the term "Mullerian inhibitory substance type I receptor" or "MISRI", also known as "AMHR1", has its general meaning in the art. ALK2, ALK3 and ALK6 are three variants of MISRI. Phosphorylation of MISRI induces SMAD1 / 5 / 8 phosphorylation, and together with SMAD4, regulates different responsive genes depending on target tissue.

[0010] As used herein, the term "ALK2," which stands for "activin receptor-like kinase 2," also known as "activin A receptor type I," has its general meaning in the art and refers to the protein encoded by the ACVR1 gene on human chromosome 2q24.1. Its entrez reference number is 90 and its Uniprot reference number is Q04771.

[0011] As used herein, "ALK3," also known as "activin receptor-like kinase 3," is also known as "bone morphogenetic protein receptor type 1A" (BMPR-1A), has its common meaning in the art, and refers to the protein encoded by the BMPR1A gene on human chromosome 10q23.2. Its entrez reference number is 657 and its Uniprot reference number is P36894.

[0012] As used herein, the term "ALK6," which stands for "activin receptor-like kinase 6," also known as "bone morphogenetic protein receptor type 1B," has its general meaning in the art and refers to the protein encoded by the BMPR1B gene on human chromosome 4q22.3. Its entrez reference number is 658 and its Uniprot reference number is O00238.

[0013] As used herein, the terms "antibody" or "immunoglobulin" have the same meaning and are used in the present invention in the same way. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies and antibody fragments. In a natural antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains a distinct sequence domain. The light chain contains two domains: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains, namely, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). Both the variable domain of the light chain (VL) and the variable domain of the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domain of the light chain (CL) and the constant domain of the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental transfer, complement binding and binding to Fc receptors (FcR). Fv fragments are the N-terminal part of the Fab fragment of immunoglobulins and consist of one light chain and one variable part of a heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is composed of residues mainly derived from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.Each of the light and heavy chains of an immunoglobulin has three CDRs, which are named L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the V regions of the heavy and light chains. Framework region (FR) refers to the amino acid sequence inserted between the CDRs.

[0014] In the context of the present invention, the amino acid residues of the antibodies of the invention are numbered according to the IMGT numbering system. The IMGT unique numbering is defined to compare variable domains, whatever the antigen receptor, chain type or species (Lefranc M.-P., “Unique database numbering system for immunogenetic analysis” Immunology Today, 18, 509 (1997); Lefranc M.-P., “The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains” The Immunologist, 7, 132-136 (1999).; Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains” Dev. Comp. Immunol., 27, 55-77 (2003). In the IMGT-specific numbering, conserved amino acids always have the same positions, e.g., cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine or tryptophan 118. The IMGT-specific numbering provides standard delimitations for the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, FR4-IMGT: positions 118-128) and the complementarity determining regions: CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, CDR3-IMGT: positions 105-117. If the length of the CDR3-IMGT is less than 13 amino acids, gaps are made from the top of the loop in the following order (111, 112, 110, 113, 109, 114, etc.).If the length of the CDR3-IMGT is greater than 13 amino acids, additional positions are created between positions 111 and 112 at the top of the CDR3-IMGT loop in the following order (112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc.) (http: / / www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html).

[0015] As used herein, the term "amino acid sequence" has its general meaning, a sequence of amino acids that gives a protein its primary structure. According to the present invention, an amino acid sequence can be modified with one, two or three conservative amino acid substitutions without significant loss of interactive binding ability. "Conservative amino acid substitution" means that an amino acid can be replaced with another amino acid that has a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0016] According to the present invention, a first amino acid sequence having at least 70% identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).

[0017] In the sense of the present invention, "identity" is calculated by comparing two aligned sequences within a comparison window. Sequence alignment makes it possible to determine the number of positions (nucleotides or amino acids) that are common to the two sequences within the comparison window. The number of common positions is then divided by the total number of positions within the comparison window and multiplied by 100 to obtain the identity percentage. The determination of the sequence identity percentage can be performed manually or with the aid of well-known computer programs.

[0018] As used herein, the terms "purified" and "isolated" in reference to an antibody or polypeptide of the invention mean that the antibody or polypeptide is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means that the antibody or polypeptide is at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and more preferably at least 98% by weight, antibody relative to the total weight of macromolecules present.

[0019] As used herein, the term "specificity" refers to an antibody that can detectably bind to an epitope present on an antigen such as MIS while having a relatively small detectable reactivity with proteins other than MIS. Specificity can be determined relatively by binding assays or competitive binding assays, for example, using a Biacore instrument, as described elsewhere herein. Specificity can be shown by the affinity / avidity ratio of binding to a specific antigen versus non-specific binding to other unrelated molecules, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or more (in this case, the specific antigen is MIS).

[0020] The term "affinity" as used herein refers to the binding strength of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of a Biacore instrument.

[0021] As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like, as used herein, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0022] As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA molecule or an RNA molecule.

[0023] antibody Sequences of interest in this application are listed in Table 1 below:

[0024] [Table 1]

[0025] A first aspect of the present invention relates to anti-Mullerian inhibiting substance (MIS) antibodies, particularly to anti-Mullerian inhibiting substance (MIS) antibodies in purified or isolated form.

[0026] Thus, the present invention relates to isolated anti-Mullerian inhibiting substance (MIS) antibodies, including: (a) a heavy chain, wherein the variable domain comprises: an H-CDR1 having the sequence set forth as SEQ ID NO:1; an H-CDR2 having the sequence set forth as SEQ ID NO:2 or SEQ ID NO:3; and an H-CDR3 having the sequence set forth as SEQ ID NO:4; and (b) A light chain ("B10 derivative"), wherein the variable domain comprises an L-CDR1 having the sequence shown as SEQ ID NO:5; an L-CDR2 having the sequence shown as SEQ ID NO:6; and an L-CDR3 having the sequence shown as SEQ ID NO:7.

[0027] Thus, the invention provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of B10. A functional variant of a VL, VH, or CDR used in the context of a monoclonal antibody of the invention allows the antibody to still retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or specificity / selectivity of the parent antibody (i.e., the B10 antibody), and in some cases, such a monoclonal antibody of the invention may associate with greater affinity, selectivity, and / or specificity than the parent Ab. Such variants have been produced by a number of techniques, including CDR mutations (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), the use of mutant strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, These affinity maturation methods can be obtained by a number of affinity maturation protocols, including those described in the literature (see, for example, the American Journal of Clinical Chemistry, 1998). Vaughan et al. (supra) discuss these affinity maturation methods. Such functional variants typically retain significant sequence identity to the parent Ab. The sequences of the CDR variants may differ from those of the CDRs of the parent antibody sequence primarily through conservative substitutions; for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more (e.g., about 65-95%, e.g., about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue substitutions.The sequences of the CDR variants may differ from those of the CDRs of the parent antibody sequence primarily through conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions may be defined by substitutions within classes of amino acids as reflected as follows: Aliphatic residues I, L, V, and M Cycloalkenyl residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in the turn are A, C, D, E, G, H, K, N, Q, R, S, and P, and residue T involved in the formation Mobile residues Q, T, K, S, G, P, D, E, and R

[0028] Further conservative substitution classifications include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation of hydrophobic / hydrophilic nature and weight / size of residues is also substantially retained in variant CDRs compared to CDRs of B10. The importance of hydropathic amino acid index in conferring interactive biological function to proteins is generally understood in the art. It is recognized that the relative hydropathicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on their hydrophobic and charge characteristics. These are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). Retention of similar residues may additionally or alternatively be measured by a similarity score as determined by use of a BLAST program (e.g., BLAST 2.2.8, available through NCBI, using standard settings of BLOSUM62, open gap=ll and extend gap=l). Suitable variants typically exhibit at least about 70% identity to the parent peptide.

[0029] Thus, in some embodiments, the isolated anti-MIS antibody comprises: (a) a variable heavy chain having at least 70% identity to the sequence set forth as SEQ ID NO:8 or SEQ ID NO:9; and (b) a variable light chain having at least 70% identity to the sequence shown as SEQ ID NO:10.

[0030] In some embodiments, the isolated anti-MIS antibody comprises: (a) a variable heavy chain having at least 70% identity to the sequence set forth as SEQ ID NO:8, wherein the variable domain comprises a VH-CDR1 having the sequence set forth as SEQ ID NO:1; a VH-CDR2 having the sequence set forth as SEQ ID NO:2; and a VH-CDR3 having the sequence set forth as SEQ ID NO:4; and (b) a variable light chain having at least 70% identity to the sequence set forth as SEQ ID NO:10, wherein the variable domain comprises a VL-CDR1 having the sequence set forth as SEQ ID NO:5; a VL-CDR2 having the sequence set forth as SEQ ID NO:6; and a VL-CDR3 having the sequence set forth as SEQ ID NO:7 ("B10 native").

[0031] In some embodiments, the isolated anti-MIS antibody comprises: (c) a variable heavy chain having at least 70% identity to the sequence set forth as SEQ ID NO:9, wherein the variable domain comprises a VH-CDR1 having the sequence set forth as SEQ ID NO:1; a VH-CDR2 having the sequence set forth as SEQ ID NO:3; a VH-CDR3 having the sequence set forth as SEQ ID NO:4; and (d) a variable light chain having at least 70% identity to the sequence set forth as SEQ ID NO:10, wherein the variable domain comprises a VL-CDR1 having the sequence set forth as SEQ ID NO:5; a VL-CDR2 having the sequence set forth as SEQ ID NO:6; and a VL-CDR3 having the sequence set forth as SEQ ID NO:7 ("B10 derivative").

[0032] In some embodiments, the isolated anti-MIS antibody comprises a variable heavy chain having the sequence shown as SEQ ID NO:8: and a variable light chain having the sequence shown as SEQ ID NO:10 ("B10 native").

[0033] In some embodiments, the isolated anti-MIS antibody comprises a variable heavy chain having the sequence shown as SEQ ID NO:9: and a variable light chain having the sequence shown as SEQ ID NO:10 ("B10 derivative").

[0034] In some embodiments, the isolated anti-MIS antibody blocks recruitment of the MIS type I receptor MISRI (ie, ALK2, ALK3, or ALK6) by the complex MISRII / MIS.

[0035] The antibodies of the present invention are produced by any technique known in the art, including, but not limited to, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, once the amino acid sequence of the desired sequence is known, one skilled in the art can easily produce said antibodies by standard polypeptide production techniques. For example, they can be synthesized using the well-known solid-phase method, preferably using a commercially available peptide synthesizer (e.g., manufactured by Applied Biosystems, Foster City, California), following the manufacturer's instructions. Alternatively, the antibodies of the present invention can be synthesized by recombinant DNA techniques well known in the art. For example, the antibodies can be obtained as DNA expression products after incorporating a DNA sequence encoding the antibody into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that will express the desired antibody, from which the antibody can be subsequently isolated using well-known techniques.

[0036] In some aspects, the antibodies of the present invention are monoclonal antibodies.

[0037] In another embodiment, the monoclonal antibody of the invention is a humanized antibody, in particular in said humanized antibody the variable domains comprise human acceptor framework regions, and optionally human constant domains (if present), and non-human donor CDRs, such as murine CDRs.

[0038] According to the present invention, the term "humanized antibody" refers to an antibody that has variable region framework and constant regions derived from a human antibody but retains the CDRs of a previous non-human antibody.

[0039] The humanized antibody of the present invention can be produced by obtaining nucleic acid sequences encoding the CDR domains as described above, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cells having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector can be either a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors, or a type in which both genes are present on the same vector (tandem type). From the viewpoints of ease of construction of the humanized antibody expression vector, ease of introduction into animal cells, and balance between the expression levels of the antibody H chain and the L chain in animal cells, the tandem type humanized antibody expression vector is preferred. Examples of the tandem type humanized antibody expression vector include pKANTEX93 (WO 97 / 10354), pEE18, etc. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985).Antibody can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)) and chain shuffling (U.S. Patent No. 5,565,332). General recombinant DNA techniques for the preparation of such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576).

[0040] In some aspects, the monoclonal antibodies of the invention are human antibodies.

[0041] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibody of the present invention may include amino acid residues that are not encoded by human immunoglobulin sequences (e.g., mutations introduced in vitro by random or site-specific mutagenesis or in vivo by somatic mutation). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are grafted onto human framework sequences.

[0042] Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368-74 (2001) and lonberg, cur. Opin.Immunol. 20; 450-459 (2008). Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech. 23; 1117-1125 (2005). Also see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSETM technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology, and U.S. Patent Application Publication US 2007 / 0061900, which describes VELOCIMOUSE® technology.The human variable region from the intact antibody produced by such animals can be further modified, for example, by combining with different human constant regions.Human antibodies can also be produced by hybridoma-based methods.Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86(1991).). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human igM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein,, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Fully human antibodies can also be derived from phage display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). Phage display technology mimics immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, followed by selection of phage by binding to an antigen of interest.One such technique is described in PCT Publication No. WO 99 / 10494.The human antibody described herein can also be prepared using SCID mice that have been reconstituted with human immune cells so that human antibody response can be generated upon immunization.Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 (Wilson et al.).

[0043] In one embodiment, the antibody of the present invention is an antigen-binding fragment selected from the group consisting of: Fab, F(ab)'2, single domain antibody, ScFv, Sc(Fv)2, diabody, triabody, tetrabody, unibody, minibody, maxibody, small modular immunopharmaceutical (SMIP), a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody as an isolated complementarity determining region (CDR), and a fragment comprising or consisting of: a VL chain and an amino acid sequence having at least 70% identity with a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:25 and SEQ ID NO:34; or a VH chain and an amino acid sequence having at least 70% identity with a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26 and SEQ ID NO:35.

[0044] The term "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., MIS). The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments that fall within the scope of the term antigen-binding fragment of an antibody include Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; Fab' fragments, which are monovalent fragments consisting of VL, VH, CL, CH1 domains and hinge region; F(ab')2 fragments, which are bivalent fragments that contain two Fab' fragments linked by disulfide bridges at the hinge region; Fd fragments, which are the VH domains of a single arm of an antibody; single-domain antibody (sdAb) fragments consisting of VH or VL domains (Ward et al., 1989 Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by an artificial peptide linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (ScFv); see, for example, Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. Bivalent and multivalent antibody fragments can be formed spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs by a peptide linker, such as bivalent sc(Fv)2. Such single-chain antibodies contain one or more antigen-binding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Unibodies are another type of antibody fragment that lacks the hinge region of an IgG4 antibody.The deletion of the hinge region results in a molecule that is essentially half the size of a conventional IgG4 antibody and has a monovalent binding region rather than the bivalent binding region of IgG4 antibodies. Antigen-binding fragments can be incorporated into single domain antibodies, SMIPs, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabody", "tribody" or "tetrabody" refers to a small antibody fragment with multiple antigen-binding sites (two, three or four), which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain, resulting in two antigen-binding sites. The antigen-binding fragment can be assembled into a single-chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0045] The Fab of the present invention can be obtained by treating an antibody that specifically reacts with MIS with the protease papain. Alternatively, the Fab can be produced by inserting DNA encoding the Fab of the antibody into a vector for a prokaryotic or eukaryotic expression system and introducing the vector into a prokaryote or eukaryote (as appropriate) to express the Fab.

[0046] The F(ab')2 of the present invention can be obtained by treating an antibody that specifically reacts with MIS with the protease pepsin. In addition, the F(ab')2 can be produced by linking the following Fab' via a thioether bond or disulfide bond.

[0047] The Fab' of the present invention can be obtained by treating F(ab')2 that specifically reacts with MIS with a reducing agent, dithiothreitol. Alternatively, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into an expression vector for prokaryotes or eukaryotes, and introducing the vector into a prokaryote or eukaryote (as appropriate) to carry out expression.

[0048] The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as described above, constructing DNA encoding the scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism (as appropriate) to express the scFv. To generate humanized scFv fragments, a well-known technique called CDR grafting may be used, which involves selecting the complementarity determining regions (CDRs) from a donor scFv fragment and grafting them into a human scFv fragment framework with a known three-dimensional structure (see, for example, WO98 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0049] A domain antibody (dAb) is the smallest functional binding unit of an antibody (molecular weight approximately 13 kDa) and corresponds to either the heavy chain variable region (VH) or the light chain variable region (VL) of an antibody. Further details regarding domain antibodies and methods for their production can be found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004 / 0110941; EP 1433846, 0368684 and 0616640; WO 2005 / 035572, 2004 / 101790, 2004 / 081026, 2004 / 058821, 2004 / 003019 and 2003 / 002609, each of which is incorporated by reference in its entirety.

[0050] Unibody is another antibody fragment technology based on the removal of the hinge region of IgG4 antibody. The deletion of the hinge region results in a molecule that is essentially half the size of conventional IgG4 antibody and has a monovalent binding region instead of a bivalent binding region. Furthermore, since unibody is almost smaller, they may show better distribution and potentially advantageous efficacy on larger solid tumors. Further details regarding unibody can be obtained by reference to WO 2007 / 059782, which is incorporated by reference in its entirety.

[0051] The antibody of the present invention can be of any isotype. The choice of isotype will typically be guided by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3 and IgG4. Either kappa or lambda of the human light chain constant region may be used. If desired, the class of the monoclonal antibody of the present invention may be switched by known methods. A typical class switching technique may be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. Thus, the effector function of the human monoclonal antibody of the present invention may be altered by switching the isotype, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody for various therapeutic applications.

[0052] In some aspects, the antibodies of the invention are full-length antibodies.

[0053] In some embodiments, the full length antibody is an IgG1 antibody.

[0054] In some embodiments, the full length antibody is an IgG4 antibody.

[0055] In some embodiments, the IgG4 antibody is a stabilized IgG4 antibody. An example of a suitable stabilized IgG4 antibody is an antibody in which the arginine at position 409 (as indicated in the EU index as in Kabat et al., supra) in the heavy chain constant region of human IgG4 is replaced with lysine, threonine, methionine or leucine, preferably lysine (described in WO2006033386), and / or an antibody in which the hinge region comprises the Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008145142, which is incorporated herein by reference in its entirety.

[0056] In some embodiments, the monoclonal antibodies of the invention are of a type other than IgG4, e.g., IgG1, IgG2 or IgG3, that have been mutated to reduce or even eliminate their ability to mediate effector functions such as ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).

[0057] A further aspect of the invention refers to cross-competing antibodies that cross-compete with the antibodies of the invention for binding to MIS.

[0058] As used herein, the term "bind" in the context of the binding of an antibody to a given antigen or epitope typically refers to a binding affinity of about 10, as determined by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, for example, using a soluble form of the antigen as the ligand and the antibody as the analyte, or vice versa. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11The affinity is to bind with an affinity corresponding to a KD of M or even lower. This affinity can be determined using other methods such as Scatchard plots using radioactive antibodies or MIS, or ELISA, or it can be evaluated by EC50. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin a series of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a KD that is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, such as at least 100,000 times lower than the KD of binding to a non-specific antigen that is not identical to or closely related to the given antigen (e.g., BSA, casein). If the KD of an antibody is very low (i.e., the antibody has high affinity), the KD at which the antibody binds to the antigen is typically at least 10,000 times lower than the KD for the non-specific antigen. An antibody is said to essentially not bind to an antigen or epitope if the binding is not detectable (e.g., using plasmon resonance (SPR) technology on a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte) or is 100-fold, 500-fold, 1000-fold or more than 1000-fold less than the binding detected by the antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

[0059] The antibody of the present invention can be assayed for specific binding by any method known in the art. Many different competitive binding assay formats can be used for epitope binding. The immunoassays that can be used include, but are not limited to, competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitin assay, gel diffusion precipitin assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay and complement fixation assay. Such assays are conventional and well known in the art (see, for example, Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York).

[0060] Additional antibodies can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the present invention in standard MIS binding assays. If a test antibody has the ability to inhibit the binding of an antibody of the present invention to MIS, it is demonstrated that the test antibody can compete with that antibody for binding to MIS; such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially close) epitope on MIS as the competing antibody. Thus, another aspect of the present invention provides antibodies that bind to the same antigen as the antibodies disclosed herein and compete with them. As used herein, an antibody "competes" for binding if the competing antibody inhibits MIS binding of an antibody or antigen-binding fragment of the invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% in the presence of an equimolar concentration of the competing antibody.

[0061] In other embodiments, the antibodies or antigen-binding fragments of the invention bind to one or more epitopes of MIS. In some embodiments, the epitopes to which the antibodies or antigen-binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the antibodies or antigen-binding fragments bind are non-linear conformational epitopes.

[0062] In some embodiments, a cross-competing antibody of the invention cross-competes for binding to MIS with an antibody comprising: (a) a heavy chain, the variable domain comprising: a VH-CDR1 having the sequence set forth as SEQ ID NO:1; a VH-CDR2 having the sequence set forth as SEQ ID NO:2 or SEQ ID NO:3; and a VH-CDR3 having the sequence set forth as SEQ ID NO:4; and (b) a light chain, wherein the variable domain comprises a VL-CDR1 having the sequence set forth as SEQ ID NO:5; a VL-CDR2 having the sequence set forth as SEQ ID NO:6; and a VL-CDR3 having the sequence set forth as SEQ ID NO:7.

[0063] In some embodiments, a cross-competing antibody of the invention cross-competes for binding to MIS with an antibody comprising a variable heavy chain having the sequence shown as SEQ ID NO:8 or SEQ ID NO:9; and a variable light chain having the sequence shown as SEQ ID NO:10.

[0064] In some embodiments, the cross-competing antibodies of the invention retain the capability of an anti-MIS antibody comprising a variable heavy chain having the sequence shown as SEQ ID NO:8 or SEQ ID NO:9; and a variable light chain having the sequence shown as SEQ ID NO:10.

[0065] The engineered antibodies of the present invention include those in which modifications have been made to framework residues in VH and / or VL, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequence to its germline configuration, the somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present invention. Another type of framework modification involves mutating one or more residues in the framework region or even in one or more CDR regions to remove a T-cell epitope, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in US Patent Publication 20030153043 by Carr et al.

[0066] In some embodiments, the glycosylation of the antibody is modified. For example, glycosylation can be altered to increase the affinity of the antibody to the antigen. Such carbohydrate modification can be achieved, for example, by modifying one or more sites of glycosylation in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen. Such an approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0067] In some embodiments, several mutations are made to amino acids located in aggregation "hot spots" in and near the first CDR (CDR1) to reduce the antibody's susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

[0068] In another embodiment, the antibody is modified to increase its biological half-life.Various approaches are possible.For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Patent No. 6,277,375 by Ward.Alternatively, to increase biological half-life, the antibody can be modified to contain salvage receptor binding epitopes in the CH1 or CL region, which are adopted from the two loops of the CH2 domain of the Fc region of IgG, as described in US Patent Nos. 5,869,046 and 6,121,022 by Presta et al. Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of IgG from mother to fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants having a substitution at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a variant having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0069] Another modification of the antibody herein envisioned by the present invention is pegylation. For example, the antibody can be pegylated to increase the biological (e.g., serum) half-life of the antibody. To pegylate the antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0070] Another modification of the antibody envisaged by the present invention is the conjugation or protein fusion of at least the antigen-binding region of the antibody of the present invention with a serum protein (such as human serum albumin or a fragment thereof) to increase the half-life of the resulting molecule. Such an approach is described, for example, in EP 0 322 094 by Ballance et al. Another possibility is the fusion of at least the antigen-binding region of the antibody of the present invention with a protein capable of binding to a serum protein (such as human serum albumin) to increase the half-life of the resulting molecule. Such an approach is described, for example, in EP 0 486 525 by Nygren et al.

[0071] Polysialylation is another technique that uses the natural polymer polysialic acid (PSA) to extend the activity time and improve the stability of therapeutic peptides and proteins. PSA is a polymer (sugar) of sialic acid. When used in drug delivery of proteins and therapeutic peptides, polysialic acid provides a protective microenvironment against conjugation. This increases the activity time of the therapeutic protein in circulation and prevents it from being recognized by the immune system. PSA polymers are found naturally in the human body. They were adopted by certain bacteria and evolved over millions of years to line the bacterial wall. These naturally polysialylated bacteria were then able to ward off the body's defense system through molecular mimicry. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with defined physical properties. Bacterial PSA is chemically identical to human PSA and is completely non-immunogenic when coupled to proteins.

[0072] Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy corn starch and can be metabolized by enzymes in the body. HES solutions are usually administered to replace missing blood volume and improve the rheological properties of blood. HESylation of antibodies allows for an increased half-life in circulation by increasing the stability of the molecule as well as by decreasing renal clearance, resulting in increased biological activity. A wide variety of HES-antibody conjugates can be customized by modifying various parameters such as the molecular weight of HES.

[0073] Nucleic Acids, Vectors, Recombinant Host Cells and Uses Thereof A further object of the invention relates to a nucleic acid molecule encoding an anti-MIS antibody according to the invention, in particular a nucleic acid molecule encoding the heavy or light chain of an anti-MIS antibody according to the invention.

[0074] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector. As used herein, the terms "vector", "cloning vector" and "expression vector" refer to a vehicle that can introduce DNA or RNA sequences (e.g., foreign genes) into host cells to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. To this end, a further aspect of the present invention relates to vectors that contain the nucleic acid of the present invention. Such vectors can include regulatory elements, such as promoters, enhancers, terminators, etc., that cause or direct the expression of the antibody when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), the immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc. Any expression vector for animal cells can be used as long as a gene encoding a human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4- (Miyaji H et al. 1990), etc. Other examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus, retrovirus, herpes virus and AAV vectors.Such recombinant virus can be produced by techniques known in the art, for example, by transfecting helper plasmid or virus into packaging cell or by transient transfection.Typical examples of virus packaging cell include PA317 cell, PsiCRIP cell, GPenv+ cell, 293 cell, etc.Detailed protocols for producing such replication-defective recombinant virus can be found, for example, in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0075] A further aspect of the present invention relates to a host cell transfected, infected or transformed with the nucleic acid and / or vector according to the invention.

[0076] A variety of expression vector / host systems can be utilized to contain and express sequences encoding peptides or proteins. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell systems infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell systems transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids; see Babe et al., 2000); or animal cell systems. Those skilled in the art are aware of various techniques for optimizing mammalian expression of proteins. See, for example, Kaufman, 2000; Colosimo et al., 2000. Mammalian cells useful in recombinant protein production include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those skilled in the art and are briefly described herein below. Mammalian host systems for expression of recombinant proteins are also well known to those skilled in the art. Host cell lines can be selected for their particular ability to process expressed proteins or to produce particular post-translational modifications that are useful in providing protein activity. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the "prepro" form of the protein can also be important for correct insertion, folding and / or function.Different host cells, such as CHO, HeLa, MDCK, 293, WI38, etc., have specific cellular and characteristic mechanisms for such post-translational activities and can be selected to ensure correct modification and processing of the introduced foreign protein.

[0077] Recombinant production of the antibodies and polypeptides of the invention will require the use of vectors that contain polynucleotide molecules that encode the antibodies and polypeptides of the invention. Methods for preparing such vectors as well as producing host cells transformed with such vectors are well known to those skilled in the art.

[0078] The selection of an appropriate expression vector for the expression of the antibody of the present invention will of course depend on the specific host cell to be used and is within the skill of the ordinary technician. Expression requires that appropriate signals are provided in the vector, such as enhancers / promoters of both viral and mammalian origin that can be used to drive the expression of the nucleic acid of interest in the host cell. Usually, the nucleic acid to be expressed is under the transcriptional control of the promoter. "Promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a gene. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding the protein of interest (e.g., a monoclonal antibody). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if it directs the transcription of that sequence.

[0079] The term "transformation" means the introduction of a "foreign" (i.e., foreign or extracellular) gene, DNA sequence, or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that accepts and expresses the introduced DNA or RNA has been "transformed."

[0080] The nucleic acids of the present invention can be used to produce the antibodies of the present invention in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions, for example, for the expression of a protein encoded by a foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and a plasmid vector, an insect host cell and a baculovirus vector, and a mammalian host cell and a vector. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces, yeast, mammalian cell lines (such as Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (such as those produced from lymphoblast cells, fibroblast cells, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the present invention, comprising the steps of (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the resulting recombinant host cell in vitro or ex vivo, and (iii) optionally selecting cells expressing and / or secreting said antibody. Such recombinant host cells can be used for the production of the antibodies of the present invention.

[0081] The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0082] Therapeutic Methods and Uses The antibody of the present invention is used as an inhibitor of MIS. The antibody of the present invention is particularly suitable for treating MIS or MISRII positive cancer.

[0083] Thus, the present invention relates to the antibody of the present invention for use in the treatment of MIS or MISRII positive cancer.

[0084] In other words, the present invention relates to a method of treating MIS or MISRII positive cancer in a subject in need thereof.

[0085] As used herein, the term "subject" refers to any mammal, such as rodent, feline, canine, primate or human.In some embodiments of the present invention, subject refers to any subject that suffers from or is likely to suffer from MIS or MISRII positive cancer.In particular, in preferred embodiments, subject is the human that suffers from or is likely to suffer from gynecological cancer, lung cancer or colorectal cancer.

[0086] In some embodiments, the subject is a human suffering from or susceptible to ovarian cancer.

[0087] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatments and curative or disease-modifying treatments, including treatments of subjects at risk of or suspected of suffering from a disease, as well as subjects who are ill or have been diagnosed with a disease or medical condition, and also includes suppression of clinical recurrence. Treatments may be administered to subjects with a medical disorder or subjects who may eventually acquire the disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or to extend the subject's survival beyond the expected survival in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, such as a pattern of dosing used during treatment. Therapeutic regimens may include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. The induction regimen may adopt (partially or entirely) a "loading regimen" that may include administering a much higher dose of drug than the dose that the physician would adopt during the maintenance regimen, administering a drug more frequently than the frequency that the physician would administer the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) that is used to maintain a subject during disease treatment, for example, to keep the subject in remission for an extended period of time (months or years). A maintenance regimen may adopt continuous therapy (e.g., administering a drug at regular intervals, for example, once a week, once a month, once a year, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching a certain predetermined criterion (e.g., pain, disease symptoms, etc.).

[0088] As used herein, "MIS or MISRII positive cancer" refers to a cancer that expresses MIS. In some embodiments, the MIS or MISRII positive cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, colorectal cancer or gynecological cancer (see Kim et al., 2014).

[0089] In some embodiments, the MIS or MISRII positive cancer is lung cancer, colorectal cancer or gynecological cancer.

[0090] As used herein, the term "lung cancer", also known as "pulmonary malignant tumor", includes the widely accepted medical definition that defines lung cancer as a medical condition characterized by uncontrolled cell growth in the tissues of the lung. The main types of lung cancer are pulmonary carcinoid tumors, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. In addition, the term "lung cancer" includes any type of lung cancer at any stage of progression. The most commonly used staging system for lung cancer is the American Joint Committee on Cancer (AJCC) TNM system, which is based on tumor size, spread to nearby lymph nodes and spread to distant sites (metastasis).

[0091] As used herein, the term "colorectal cancer" or "CRC" includes the widely accepted medical definition that defines colorectal cancer as a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). In addition, as used herein, the term "colorectal cancer" also includes medical conditions characterized by cancer of cells of the duodenum and small intestine (jejunum and ileum). In addition, the term "colorectal cancer" includes any type of colorectal cancer at any stage of progression. The earliest stage of colorectal cancer is called stage 0 (very early and superficial cancer), which then varies from stage I to IV. In stage IV colorectal cancer, also known as metastatic colorectal cancer, the cancer has spread beyond the colon or rectum to distant organs such as the liver or lungs. The most commonly used staging system for CRC is the American Joint Committee on Cancer (AJCC) TNM system, which is based on tumor size, spread to nearby lymph nodes, and spread to distant sites (metastasis).

[0092] As used herein, the term "gynecological cancer" has its general meaning in the art and refers to cancer that occurs in the female reproductive organs. Types of gynecological cancer are uterine cancer, also known as cervical cancer, womb cancer or endometrial cancer, ovarian cancer, vaginal cancer, vulvar cancer, primary peritoneal cancer, gestational trophoblastic disease and fallopian tube cancer. Cervical cancer occurs when cells of the cervix grow abnormally and invade other tissues and organs of the body, and includes squamous cell carcinoma; adenocarcinoma; adenosquamous cell carcinoma; small cell carcinoma: neuroendocrine tumor; ground glass cell carcinoma; trophoblastic tubular adenocarcinoma; cervical melanoma and cervical lymphoma. Uterine cancer refers to any type of cancer that occurs in the uterus, including endometrial carcinoma, such as endometrial adenocarcinoma, endometrial adenosquamous carcinoma, serous papillary carcinoma, uterine clear cell carcinoma, endometrial mucinous carcinoma, endometrial mucinous adenocarcinoma, and endometrial squamous cell carcinoma; endometrial transitional cell carcinoma; endometrial stromal sarcoma; malignant mixed Müllerian tumor; uterine fibroids; and uterine sarcomas, such as uterine carcinosarcoma, uterine adenosarcoma, and uterine leiomyosarcoma. Vaginal cancer is a rare cancer that occurs in the vagina, including vaginal squamous cell carcinoma; vaginal melanoma; and vaginal sarcoma. Vulvar cancer is a type of cancer that occurs in the external surface area of ​​the female genital tract, including vulvar squamous cell carcinoma; vulvar melanoma; vulvar basal cell carcinoma; Bartholin's gland carcinoma; vulvar adenocarcinoma, and vulvar sarcoma. Ovarian cancer is cancer that forms in or on the ovaries and includes ovarian epithelial tumors, such as ovarian mucinous carcinoma, high-grade serous carcinoma, ovarian endometrioid carcinoma, ovarian clear cell carcinoma, ovarian low malignant potential tumors, and primary peritoneal carcinoma; germ cell tumors, such as teratomas, dysgerminomas, ovarian germ cell carcinomas, choriocarcinoma tumors, and endodermal sinus tumors; sex cord-stromal tumors, such as granulosa cell tumors, granulosa-thecal tumors, ovarian fibromas, Leydig cell tumors, Sertoli cell tumors, Sertoli-Leydig tumors, and ginandroblastomas; ovarian sarcomas, such as ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, and ovarian fibrosarcoma; Krukenberg tumors; and ovarian cysts.

[0093] In some embodiments, the MIS or MISRII positive cancer is ovarian cancer.

[0094] As used herein, "therapeutically effective amount" refers to the minimum amount of active agent required to provide a therapeutic benefit to a patient. For example, a "therapeutically effective amount of an active agent" to a patient is an amount of active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression or physical condition associated with a disease affecting the patient.

[0095] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an antibody of the present invention) to a subject, for example, by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is treated, administration of the substance is typically performed after the onset of the disease or a symptom thereof. When a disease or a symptom thereof is prevented, administration of the substance is typically performed before the onset of the disease or a symptom thereof.

[0096] The antibodies of the invention can be administered in combination with conventional treatments for MIS or MISRII positive cancers.

[0097] Therefore, the present invention also refers to i) an anti-Müllerian inhibitory substance antibody according to the invention and ii) a conventional treatment for use in the treatment of MIS or MISRII positive cancers.

[0098] In other words, the present invention refers to a method of treating a MIS or MISRII positive cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-MIS antibody of the present invention and a conventional treatment for the MIS or MISRII positive cancer.

[0099] As used herein, the term "conventional treatment" refers to any compound, natural or synthetic, used to treat MIS or MISRII positive cancers.

[0100] In certain embodiments, conventional treatment refers to radiation therapy, immunotherapy, or chemotherapy.

[0101] According to the present invention, compounds used in the conventional treatment of MIS or MISRII positive cancers may be selected from the group consisting of EGFR inhibitors such as cetuximab, panitumumab, bevacizumab and ramucirumab; kinase inhibitors such as erlotinib, gefitinib, afatinib, regorafenib and larotrectinib; immune checkpoint inhibitors; chemotherapeutic agents and radiotherapeutic agents.

[0102] As used herein, the term "chemotherapy" refers to cancer treatment using one or more chemotherapeutic agents.

[0103] As used herein, the term "chemotherapeutic agent" refers to a chemical compound that is effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins, particularly bullatacin and bullatacinone; camptothecin, including synthetic analogs topotecan and irinotecan; bryostatin; kallistatin; CC-1065, including its adozelesin, carzelesin and biceresin synthetic analogs; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); erytherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g. calicheamicins, especially calicheamicin 111 and calicheamicin 211, e.g. Agnew See Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemycins, including dynemycin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramicin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin n), 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfilomycin in), puromycin, quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioglucose, anine;pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, trifluridine, tipiracil, enocitabine, floxuridine, 5-FU;androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;antiadrenal agents such as aminoglutethimide, mitotane, trilostane;folic acid supplements such as frolinic acid;aceglatone;aldophospharnide glycoside;aminolevulinic acid;amsacrine;bestrabucil;bisantrene;edatraxate;defo famine;Demecolcine;Diaziquone;Elfornithine;Elliptinium acetate;Epothilones;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Maytansinoids, such as maytansine and ansamitocins;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Fenamet;Pirarubicin;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK (registered trademark);Razoxane;Rhizoxin;Sizofiran;Spirogennanium;Tenuazonic acid;Triaziquone;2 ,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A, roridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum, such as oxaliplatin, cisplatin and carboplatin; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1; topoisomerase inhibitor RF S2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; ziv-aflibercept;and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens (including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (Fareston)); and antiandrogens (for example, flutamide, nilutamide, bicalutamide, leuprolide, and goserelin); and pharmaceutically acceptable salts, acids or derivatives of any of the above.;

[0104] "Pharmaceutically" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, particularly a human (as appropriate). A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0105] As used herein, the term "radiotherapy" has its general meaning in the art and refers to the treatment of MIS or MISRII positive cancers with ionizing radiation. Ionizing radiation stores energy that injures or destroys cells in the treated area (target tissue) by damaging their genetic material, making these cells unable to continue growing. One commonly used form of radiation therapy involves photons, such as X-rays. Depending on the amount of energy they carry, the rays can be used to destroy cancer cells on the surface or deeper in the body. The higher the energy of the X-ray beam, the deeper the X-rays can penetrate into the target tissue. Linear accelerators and betatrons produce X-rays with greater energy. Using a machine to focus radiation (such as X-rays) on the colorectal cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays occur naturally as certain elements (such as radium, uranium, and cobalt 60) emit radiation as they break down or decay. In some embodiments, the radiation therapy is external beam radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT) (delivering shaped beams from different directions to closely fit the shape of the tumor); intensity-modulated radiation therapy (IMRT), such as helical tomotherapy (shaping the radiation beam to closely fit the shape of the tumor and also varying the radiation dose according to the shape of the tumor); conformal proton beam radiation therapy; image-guided radiation therapy (IGRT) (combining scanning and irradiation techniques to provide a real-time image of the tumor and guide the radiation therapy); intraoperative radiation therapy (IORT) (delivering radiation directly to the tumor during surgery); stereotactic radiosurgery (delivering large, precise doses of radiation to a small tumor area in a single session); hyperfractionated radiation therapy, such as sequential hyperfractionated accelerated radiation therapy (CHART), (subjects are given multiple radiation therapy treatments (fractions) per day); and hypofractionated radiation therapy (a larger dose of radiation therapy per fraction is given in fewer fractions).

[0106] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with or modulates one or more immune checkpoint proteins, either in whole or in part.

[0107] As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to molecules that enhance (stimulatory checkpoint molecules) or attenuate (inhibitory checkpoint molecules) signals expressed by T cells.

[0108] Examples of stimulatory checkpoints include CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3, and VISTA.

[0109] According to the present invention, MIS inhibitors and conventional treatments can be used as a combination treatment.

[0110] As used herein, the term "combination treatment," "combination therapy," or "combination therapy" refers to a treatment using more than one pharmaceutical agent. The combination therapy may be a two-drug therapy or dual therapy. The pharmaceutical agents used in the combination treatment of the present invention are administered to a subject simultaneously, separately, or sequentially.

[0111] As used herein, the term "administration at the same time" refers to administration of two active ingredients by the same route at the same time or substantially the same time. The term "administration separately" refers to administration of two active ingredients by different routes at the same time or substantially the same time. The term "administration sequentially" refers to administration of two active ingredients at different times, with the same or different administration routes.

[0112] Pharmaceutical Compositions The anti-MIS antibodies of the invention may be used or formulated in pharmaceutical compositions.

[0113] In one aspect, the invention relates to a pharmaceutical composition comprising an anti-MIS antibody of the invention and a pharma- ceutically acceptable carrier for use in the treatment of MIS or MISRII positive cancer.

[0114] In some embodiments, the MIS or MISRII positive cancer is selected from the group consisting of gynecological cancer, lung cancer and colorectal cancer.

[0115] Typically, the inhibitors of the present invention will be combined with pharma- ceutically acceptable excipients, and optionally sustained release matrices, such as biodegradable polymers, to form a therapeutic composition.

[0116] As used herein, the terms "pharmaceutical" or "pharmaceutical acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, particularly a human (as appropriate). A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0117] In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient can be administered to animals and humans alone or in combination with another active ingredient in unit dosage form, in admixture with conventional pharmaceutical supports. Suitable unit dosage forms include oral route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0118] Preferably, the pharmaceutical composition contains a pharma- ceutically acceptable vehicle for injectable preparations. These may be, in particular, isotonic sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc., or mixtures of such salts), or dry, especially lyophilized compositions (optionally allowing the constitution of solutions for injection by adding sterile water or saline).

[0119] Suitable dosage forms for injection use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0120] Solutions containing the inhibitors of the present invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0121] The inhibitor of the present invention can be formulated into compositions in neutral or salt form.Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.Also, the salts formed with free carboxyl groups can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and from organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0122] The carrier can also be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using an absorption-delaying agent, for example, aluminum monostearate and gelatin, in the composition.

[0123] Sterile injectable solution is prepared by adding the required amount of active compound to a suitable solvent with various other ingredients as listed above, as necessary, followed by filtration sterilization.Generally, dispersion is prepared by adding various sterilized active ingredients to a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder from the previously sterile filtered solution of active ingredient and any additional desired ingredient.

[0124] Once formulated, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like can also be utilized.

[0125] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.In this regard, the available sterile aqueous medium will be known to those skilled in the art in light of this disclosure.Some variation in dosage will necessarily occur depending on the condition of the subject being treated.In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0126] In addition to the MIS inhibitors of the present invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solid forms for oral administration; liposomal formulations; sustained release capsules; and any other form currently in use.

[0127] The pharmaceutical composition of the present invention may contain any additional active agent used in the treatment of cancer selected from the group consisting of gynecological cancer, lung cancer or colorectal cancer.

[0128] In one aspect, the additional active agents may be contained in the same composition or administered separately.

[0129] In another embodiment, the pharmaceutical composition of the present invention relates to a combined preparation for simultaneous, separate or sequential use in the treatment of MIS or MISRII positive cancer.

[0130] In some embodiments, the MIS or MISRII positive cancer is selected from the group consisting of gynecological cancer, lung cancer, or colorectal cancer.

[0131] The present invention also provides a kit comprising an anti-MIS antibody of the present invention. The kit containing the anti-MIS antibody of the present invention is useful in therapeutic methods.

[0132] The present invention will be further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0133] [Figure 1] Graphical abstract of the paradoxical response of Müllerian inhibitory substance (MIS) in ovarian malignancies and the proposed therapeutic strategy of MIS inhibition. [Figure 2A] Recombinant MIS (LRMIS) induces MIS signaling in COV434-MISRII and SKOV3-MISRII cells. A. Incubation with 1.6-25 nM LRMIS for 6 h promotes apoptosis (caspase 3 / 7 activity). B. After 11 days of culture in the presence of 1.6-2.5 nM LRMIS, clonogenic survival was quantified by direct clone counting (COV434-MISRII cells) or by estimating clone numbers by OD at 595 nm after cell lysis (SKOV3-MISRII cells). [Figure 2B] Recombinant MIS (LRMIS) induces MIS signaling in COV434-MISRII and SKOV3-MISRII cells. A. Incubation with 1.6-25 nM LRMIS for 6 h promotes apoptosis (caspase 3 / 7 activity). B. After 11 days of culture in the presence of 1.6-2.5 nM LRMIS, clonogenic survival was quantified by direct clone counting (COV434-MISRII cells) or by estimating clone numbers by OD at 595 nm after cell lysis (SKOV3-MISRII cells). [Diagram 3]Involvement of ALK2, ALK3 and ALK6 in MIS action in COV434-MISRII and SKOV3-MISRII cells. Apoptosis initiation (caspase 3 / 7 activity) was analyzed after 6 h incubation of siALK2-, siALK3- or siALK6-transfected COV434-MISRII or SKOV3-MISRII cells with 25 nM MIS (starting 48 h after siRNA transfection). [Figure 4] Anti-MIS antibody B10 induces growth inhibition in COV434-MISRII, SKOV3-MISRII, OVCAR8 and KGN cells. Clonogenic survival in COV434-MISRII cells (direct clonogenic counting) and SKOV3-MISRII, OVCAR8 and KGN cells (measurement of cell confluency using a Celigo Imaging System) after 11 days of incubation with or without 333 nM B10. [Figure 5A] Anti-MIS antibody B10 induces growth inhibition in tumor cells from ovarian carcinoma ascites samples. A. Cell growth inhibition after 48 h incubation with or without (NT) 330 nM B10 (cell confluency measured with a Celigo Imaging System). B. Apoptosis induction (caspase 3 / 7 activity) after incubation with or without increasing concentrations of B10. [Figure 5B] Anti-MIS antibody B10 induces growth inhibition in tumor cells from ovarian carcinoma ascites samples. A. Cell growth inhibition after 48 h incubation with or without (NT) 330 nM B10 (cell confluency measured with a Celigo Imaging System). B. Apoptosis induction (caspase 3 / 7 activity) after incubation with or without increasing concentrations of B10. [Figure 6A]Anti-AMH antibody B10 reduces COV434-MISRII tumor growth in vivo. Nude mice bearing COV434-MISRII cell tumors were treated twice weekly for 4 weeks with B10 (anti-AMH antibody), 12G4 (anti-AMHRII antibody) (both at 10 mg / kg / injection), or vehicle (NaCl; control). A. Tumor growth curves (mean + 95% confidence interval), B. Kaplan-Meier survival curves (proportion of mice with tumor volume less than 1,500 mm3 as a function of time after implantation). [Figure 6B] Anti-AMH antibody B10 reduces COV434-MISRII tumor growth in vivo. Nude mice bearing COV434-MISRII cell tumors were treated twice weekly for 4 weeks with B10 (anti-AMH antibody), 12G4 (anti-AMHRII antibody) (both at 10 mg / kg / injection), or vehicle (NaCl; control). A. Tumor growth curves (mean + 95% confidence interval), B. Kaplan-Meier survival curves (proportion of mice with tumor volume less than 1,500 mm3 as a function of time after implantation). EXAMPLES

[0134] Materials & Methods cell line Human COV434 (sex cord stromal tumor) (Chan-Penebre et al., 2017; Zhang et al., 2000) and KGN (granulosa cell tumor) (Nishi et al., 2001) cell lines were kindly provided by PI Schrier (Department of Clinical Oncology, Leiden University Medical Center, Netherlands) and T Yanase (Kyushu University, Fukuoka, Japan), respectively. Human epithelial ovarian cancer cell lines SKOV3 and NIH-OVCAR8 were obtained from ATCC (ATCC® HTB-77) and Division of Cancer Treatment and Diagnosis, NCI, Frederick, MD, USA, respectively. Cells were grown in red phenol-free DMEM F12 medium containing 10% heat-inactivated fetal bovine serum (FBS). COV434-MISRII and SKOV3-MISRII cells were supplemented with 0.33 mg / ml geneticin (InvivoGen, ant-gn-1). The cells were incubated at 5% CO 2 The cells were grown at 37°C in a humidified atmosphere with medium changed twice weekly. Cells were harvested using 0.5mg / ml trypsin / 0.2mg / ml EDTA. All culture media and supplements were purchased from Life Technologies. Inc. (Gibco BRL). HEK293K cells (used for antibody production by the GenAc platform at IRCM) were grown in DMEM F12 with phenol red containing 10% heat-inactivated FBS.

[0135] COV434-MISRII and SKOV3-MISRII cell lines were generated by transfection of cDNA encoding full-length human MISRII (Kersual et al., 2014). cDNA encoding full-length human MISRII in pCMV6 plasmid was a generous gift by J Teixeira (Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School). First, MISRII cDNA was subcloned into pcDNA3.1.myc-His vector (Invitrogen) using EcoRI and XhoI restriction sites (enzymes from New England BioLabs) and then into pIRES1-EGFP vector (gift from F Poulat (IGH-UPR1142 CNRS)) using EcoRI and SalI sites. 24 h prior to transfection, COV434 cells were seeded at 80% confluency in 10 cm cell culture dishes. The MISRII construct was transfected using Fugene transfection kit according to the manufacturer's protocol. After 48 hours, the transfection medium was replaced with fresh medium containing 0.5mg / ml geneticin, and then replaced twice a week for 2 weeks. The cells were then harvested and sorted into 96-well plates using a FACSAria cytometer (Becton Dickinson). For each cell line, clones that strongly expressed MISRII were selected and named COV434-MISRII and SKOV3-MISRII.

[0136] Primary tumor cells from the patient's ascites Ascites samples from two ovarian cancer patients were obtained from the "Institut Cancer Montpellier, ICM" after informed consent, in accordance with French law. These two patients were selected because they had not received any chemotherapy to date and were awaiting surgical intervention at the ICM-Val d'Aurelle Hospital. Freshly obtained ascites was divided into 50 ml conical centrifuge tubes and spun at 1300 rpm for 5 min. The cell pellet was dissolved in ammonium chloride potassium buffer (ACK lysis buffer: NH 4 Cl 150 nM; KHCO 3 10nM;Na 2 Red blood cells (RBCs) were lysed for 5 min on ice by resuspending in EDTA 0.1 nM. This process was repeated until RBC lysis was complete. The cell pellet was then plated in a 150 mm cell culture dish containing 20 ml DMEM F12-Glutamax (Gibco) and 10% FBS. On the same day, 100,000 cells were harvested and MISRII expression was assessed by FACS. The cells were then plated in DMEM F12 / 10% FBS for 30 min to quickly remove adherent fibroblasts (O Donnell et al., 2014). Non-adherent cells were transferred to a new dish containing DMEM F12 / 10% FBS. Low passage cells were used for experiments or frozen in liquid nitrogen.

[0137] Mullerian inhibiting substance (MIS) production and assay Active recombinant MIS (LRMIS) described in the work by D Pepin et al. (Pepin et al., 2013, 2015) was used in our study. It contains (i) the 24AA leader sequence of albumin replacing the MIS leader sequence, which increases production and secretion, and (ii) the RARR / S Furin / kex2 consensus site replacing the native MISRAQR / S sequence at positions 423-428, which improves cleavage. Dosage determination of MIS was performed using the Elecsys® AMH (anti-Mullerian hormone) assay from Roche. As bovine MIS can signal through human MISRII (Cate et al., 1986), all experiments involving LRMIS were performed in culture medium containing 1% FBS. In these experimental conditions, endogenous MIS concentrations ranged from 5-10 pM (in fresh medium) to approximately 10-15 pM (after 5 days of cell culture). To determine the endogenous MIS concentration in cell culture supernatants, 1 million cells were plated in 10 ml DMEM F12 / 1% FBS in a 100 mm cell culture dish. Every 24 hours, 300 μl of medium was removed for MIS titration.

[0138] siRNA transfection and assay siRNA sequences were designed with the Rosetta algorithm, which is supported by Sigma-Aldrich predesigned siRNA guarantee. We used a pool of three siRNAs for each ALK receptor and MIS. Cells were plated in 24-well plates at a maximum confluency of 60-80%. Transfections were performed in medium containing 1% FBS using Lipofectamine RNAiMax transfection reagent (Thermofisher cat# 13778-150) diluted in Opti-MEM medium according to the supplier. siRNAs were diluted to 300ng / ml (siRNAs against ALK2, ALK3 and ALK6) and 1μg / ml (siRNA against MIS) in Opti-MEM, and the siRNA-Lipofectamine (1:1) mixture was added to the cells for 6 hours. Cells were washed and cultured in DMEM F12 / 1% FBS. Experiments with siRNA-transfected cells were performed 24 hours (COV434-MISRII cells) and 48 hours (SKOV3-MISRII cells) after transfection.

[0139] Development and production of anti-MIS B10 antibodies Three anti-MIS human scFv antibodies were selected by phage display after sequential panning with the Ala453-Arg560 MIS cTER domain (R&D) from the human scFv phage display library Husc I (Philibert et al., 2007; Robin and Martineau, 2012). Antibodies were first expressed in murine Ig2Ga format. MAb B10 displayed the best binding to full-length MIS as judged by ELISA (Pepin et al., 2013) and was therefore chosen for further experiments.

[0140] MAb B10 antibody was produced in HEK293T cells (ATCC CRL1573). HEK293T cells were cultured at 150 mm 2The cells were grown in 1000 ml dishes at a maximum confluency of 70%. A 1:1 mixture of 30 μg of plasmid encoding B10 and 240 μg of the transfection agent polyethyleneimine PEI (Polyscience) was kept at room temperature for 10 min and then added to the cells for 6 h. The transfection medium was then replaced with DMEM without FBS. After 5 days, the supernatant was collected, diluted (1:1) with 40 mM sodium phosphate buffer (pH 8), filtered through a 0.22 μm filter, and purified on a 1 ml Protein A column for 24 h. The antibody was eluted at acidic pH (glycine, pH 3) and immediately stabilized in Tris buffer (pH 9). The antibody was concentrated in PBS using a Centricons filter with a cut-off of 50 kDa. 200 ml of cell culture yielded approximately 1 mg of purified antibody.

[0141] Western blot analysis Cells were washed with PBS and immediately disrupted in RIPA lysis buffer (Santa Cruz) containing 200 mM PMSF solution, 100 mM sodium orthovanadate solution and protease inhibitor cocktail. Protein concentration was determined using a BCA assay protein quantification kit (Interchim). Cell extracts were heated at 95°C for 5 min, separated (50 μg protein / well) on 10% SDS-PAGE under reducing conditions (5% 2β-mercaptoethanol) and transferred to PVDF membranes (Biorad). Membranes were saturated in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk and probed with the relevant primary antibodies for 1 h at RT. After washing, peroxidase-conjugated IgG secondary antibodies (1 / 10,000) were added for 1 h at RT. After washing, antibody-antigen interactions were detected using a chemiluminescent substrate (Merck). To confirm equal loading, immunoblots were also probed with anti-GAPDH monoclonal antibody (Cell Signaling).

[0142] MIS pathway analysis Cells were cultured overnight in DMEM F12 / 1% FBS medium and then incubated with LRMIS (0–25 nM) for 6 h at 37°C. Western blotting was performed overnight at 4°C using anti-phosphorylated SMAD1 / 5, anti-phosphorylated AKT, anti-cleaved caspase 3, anti-cleaved PARP, and anti-GAPDH primary antibodies (1:1.000; Cell Signaling), anti-ALK2, and anti-ALK3 antibodies (1 μg / ml; R&D system), followed by anti-rabbit and anti-goat IgG HRP secondary antibodies (1:10.000; Sigma) for 1 h at room temperature.

[0143] Clonogenic survival Cells were plated overnight in DMEM F12 / 1% FBS medium in 24-well plates (50 cells / well). LRMIS (0–25 nM) or anti-MIS Mab B10 (333 nM) was then added for 11 days of culture. For COV434-MISRII cells growing as clearly individualized clones, colonies were fixed with a methanol / acetic acid solution (3:1) for 20 min at 4 °C, stained with 10% Giemsa, and counted. For SKOV3-MISRII, OVCAR8, KGN cells and cells derived from patient ascites, the number of clones was estimated from the confluency area determined using a Celigo Imaging System after staining the cells with Hoechst 33342 trihydrochloride (Invitrogen H1399, 0.25 μg / ml for 15 min).

[0144] Apoptosis assay Apoptosis initiation was measured using the Caspase-Glos-3 / 7 assay (Promega). Cells were plated in white 96-well plates and incubated with LRMIS (0–25 nM) for 6 h. Upon addition of the proluminescent caspase-3 / 7 DEVD-aminoluciferin substrate, caspase-3 / 7 generates free aminoluciferin that is consumed by luciferase, generating a luminescent signal proportional to caspase-3 / 7 activity. Thirty minutes after substrate addition, the luminescent signal was quantified on a PHERASTAR microplate reader.

[0145] For a more complete analysis of apoptosis, the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter IM3614) was used. Approximately 100,000 cells per well were seeded in 24-well plates and incubated with or without 50 μg / ml Mab B10, 25 nM LRMIS, or 150 nM staurosporine (positive control) for 24 h. Adherent and detached cells were collected and centrifuged at 900 rpm for 5 min. After washing with PBS, cells were stained with 130 μl of a mixture containing 10 μl of FITC-labeled Annexin V and 20 μl of 7AAD in 100 μl of Annexin buffer for 15 min on ice in the dark. After adding 400 μl of Annexin buffer, the fluorescent signal data were acquired within 30 min by flow cytometry, and the data were analyzed with Kaluza Flow Analysis software (Beckman Coulter).

[0146] Immunofluorescence For each assay, 30,000 cells were grown overnight on 22 mm square glass coverslips in DMEM F12 / 10% FBS in 35 mm culture dishes. Cells were then starved for 24 h in 1% FBS medium before being incubated with 25 nM LRMIS for 1 h 30 min. Cells were then fixed for 20 min in 3.7% paraformaldehyde / PBS and permeabilized for 30 s in acetone at -20°C. Cells were washed twice with PBS / 0.1% BSA and incubated with P3X63 (irrelevant antibody) (Kohler et al., 1976), anti-MISRII 12G4 and anti-ALK2, anti-ALK3, anti-ALK6 (R&D) primary antibodies for 1 h in the dark. After further washing, cells were incubated with goat-FITC-labeled secondary antibodies in PBS / 0.1% BSA for 1 h. They were then washed three times with PBS / 0.1% BSA and once with PBS. Coverslips were mounted with DAPI (Biotium, Inc., Fremont, Calif.) using EverBrite™ Hardest Mounting and later analyzed on a Zeiss Axioplan 2 Imaging microscope.

[0147] Cell viability assay For cell viability / proliferation testing, the CellTiter 96 AQueous One Solution cell proliferation assay system (Promega) was used according to the manufacturer's instructions. Five thousand cells were plated in each well of a 96-well plate and cultured overnight in 50 μl of DMEM F12 / 1% FBS medium. The cells were then incubated with LRMIS (0–25 nM) or anti-MIS B10 antibody (0–333 nM) for 3 days. Then, 10 μl of CellTiter 96 AQueous One Solution reagent was added per well and the plate was incubated at 37 °C in a humidified 5% CO 2 Positive control wells were incubated at room temperature until they turned brown (1-2 hours depending on cell line). Absorbance was then measured at 490 nm using a PHERASTAR microplate reader. Three replicate wells were used per condition.

[0148] Elisa Assay ELISA was used to determine the EC50 of the B10 antibody. Polyclonal anti-AMH antibody (Abcam ab 84952) was coated overnight on a 96-well high protein binding capacity plate (Nunc MaxiSorp). The plate was then washed three times and saturated with a PBS-Tween 0.01%-BSA 2% solution for 2 h. After each step, the plate was washed three times with PBS-Tween 0.01%. Recombinant AMH (25 nM) was added and incubated for 2 h at 37 °C. Antibody B10 (666-0 nM) was then added and incubated for 1 h 30 min at 37 °C. A secondary anti-Fc mouse peroxidase (HRP) antibody was incubated for 30 min and the substrate enzyme (Thermofisher, TMB) was added. The enzymatic reaction was stopped by the addition of sulfuric acid, after which the absorbance was read at 450 nm.

[0149] In vivo studies using ovarian cancer cell xenografts All animal experiments were performed in accordance with the French government guidelines for experimental animal research and the Inserm ordinance (agreement D34-172-27). For all in vivo experiments, on day 0 (D0), mice were cultured with 7.10 ng / mL BD Matrigel in a volume of 150 μl. 6 Human COV434-MISRII cells39 (1:1 ratio) were implanted subcutaneously (sc) into the right flank of female athymic nude Hsd mice (6–8 weeks old) (ENVIGO, France). Tumor volumes were 60–150 mm3 at D12–D13. 3 Mice were randomized when tumor size reached 100 mg / kg (n=5-7 mice / group). All treatments were performed by intraperitoneal (ip) injection twice weekly for 4 weeks. Anti-AMH MAb B10 (IgG2a format, produced in HEK296T cells) and anti-AMHRII MAb 12G4 (chimeric IgG1 format, produced in CHO cells) were injected at 10 mg / kg. The untreated group received saline solution (vehicle). Tumor dimensions were measured weekly with calipers and calculated using the formula: D 1 ×D 2 ×D 3 Tumor volume was calculated using the 1,500 mm2 / 2 method. 3Results were expressed by fitted Kaplan-Meier survival curves using the time required for 50% of mice to reach a volume of 1,500 mm. 3 Median survival was defined as the time a patient presented with tumor.

[0150] statistical analysis Statistical analysis of differences in caspase-3 / 7 activity and cell viability / proliferation was performed using Prism software and ANOVA (Tukey's multiple comparison test).

[0151] A linear mixed regression model was used to determine the relationship between tumor growth and days after implantation. The fixed part of the model included variables corresponding to days after implantation and the different groups. Interaction terms were incorporated into the model. Random intercepts and random slopes were included to account for time effects. Model coefficients were estimated by maximum likelihood and considered significant at the 0.05 level. 3 Survival rates were estimated using the Kaplan-Meier method until the day the tumor volume reached 100%. Median survival times are presented with 95% confidence intervals. Survival curves were compared using the log-rank test. Statistical analysis was performed using STATA 16.0 software (StataCorp, College Station, TX).

[0152] result Recombinant MIS induces MIS signaling in COV434-MISRII and SKOV3-MISRII cells Before evaluating the involvement of different MISRIs, we analyzed MIS / MISRII signaling in two MISRII-positive ovarian cancer cell lines: COV434-MISRII (Kersual et al., 2014) and SKOV3-MISRII cells. Indeed, we and other authors found that MISRII expression in cell lines derived from ovarian malignant tumors and ovarian malignant tumor ascites rapidly and progressively decreased after long-term culture (Estupina et al., 2017; Pepin et al., 2015), thus limiting experimental reproducibility. In all of the experiments described in this study, we used human recombinant AMH (LR-AMH;

[10] ) produced in CHO cells (Evitria AG, Zurich, Switzerland) according to patent WO2014 / 164891 (data not shown). LR-AMH has the advantage of being a full-length hormone, yet completely truncated, and therefore efficient and stable (Pepin et al., 2013; Wilson et al., 1993). Since it was reported that bovine AMH can signal through human AMHRII (Cate et al., 1986), we performed all experiments with LR-AMH in culture medium containing 1% FBS. In these experimental conditions, the AMH concentration in the medium ranged from 5-10 pM (in fresh medium) to approximately 10-15 pM (after 5 days of culture).

[0153] In both cell lines, SMAD1 / 5 phosphorylation was induced at all tested LRMIS concentrations (1.6–25 nM). Apoptosis, assessed by measuring caspase-3 / 7 activity, was significantly induced at 12.5 nM LRMIS in COV434-MISRII cells and at 6.3 nM LRMIS in SKOV3-MISRII cells (Figure 2A). We confirmed apoptosis induction by Western blot analysis of cleaved caspase-3 / 7 and cleaved PARP (data not shown). Furthermore, flow cytometry analysis showed that incubation with 25 nM LRMIS for 24 h strongly induced apoptosis in COV434-MISRII cells compared to untreated cells (12.5% ​​vs. 3.6% Annexin V positive cells, and 16.3% vs. 5.3% Annexin V / 7AAD positive cells), and to a lesser extent in SKOV3-MISRII cells (4.5% vs. 5.4% Annexin V positive cells, and 11.3% vs. 1.7% Annexin V / 7AAD positive cells) (data not shown). Finally, clonogenic survival was reduced in both cell lines at all tested LRMIS concentrations (Figure 2B). These results confirmed that COV434-MISRII and SKOV3-MISRII cells are valid models for studying MIS signaling.

[0154] In ovarian cancer cells, ALK3 is a major MISRI involved in MIS signaling To analyze the involvement of MISRI in MIS signaling in ovarian cancer cells, we transfected siRNAs targeting ALK2, ALK3 and ALK6 into COV434-MISRII and SKOV3-MISRII cells. Due to the role of these receptors in different signaling pathways, their shRNA-mediated silencing was lethal in these cells. PCR and Western blot analysis showed that a mixture of three siRNAs against ALK2 (siAlk2) and a mixture of three siRNAs against ALK6 (siAlk6) efficiently inhibited their expression (data not shown). Conversely, ALK3 silencing (siAlk3) was less efficient, especially in COV434-MISRII cells. Incubation with LRMIS (25 nM, 6 h) induced SMAD1 / 5 phosphorylation in siAlk2 and siAlk6 COV434-MISRII and SKOV3-MISRII cells, but not in siAlk3 COV434-MISRII and SKOV3-MISRII cells (data not shown). There was no significant difference in caspase-3 / 7 activity and cleavage between siAlk2 and siAlk6 COV434-MISRII and SKOV3-MISRII cells and those transfected with control siRNA (Figure 3). Conversely, apoptosis was reduced by approximately 25% in siAlk3 COV434-MISRII and SKOV3-MISRII cells compared to controls. These results were confirmed by Western blot analysis of PARP and caspase-3 / 7 cleavage (data not shown). These findings indicate that MIS signaling is reduced primarily in siAlk3 COV434-MISRII and SKOV3-MISRII cells despite incomplete silencing, demonstrating that ALK3 is the preferred MISRI receptor for MIS signaling in ovarian cancer cells.

[0155] MIS modulates ALK2 and ALK3 expression in ovarian cancer cells We then investigated the effect of MIS on MISRII, ALK2, ALK3 and ALK6 expression in four MISRII-positive ovarian cancer cell lines: COV434-MISRII (sex cord stromal tumor), SKOV3-MISRII (epithelial cancer), OVCAR8 (epithelial cancer), and KGN (granulosa cell tumor). Immunofluorescence (IF) analysis showed that MISRII and ALK2 were clearly expressed in all four cell lines in basal conditions (1% FBS corresponding to 10 pM MIS), and their expression was not modulated by incubation with 25 nM LRMIS for 90 minutes (data not shown). ALK3 expression was not detectable by IF in basal conditions, but was induced in all four cell lines by MIS addition (data not shown). ALK6 was not detectable in both experimental conditions.

[0156] To determine the role of ALK2 and ALK3, we then assessed their expression and that of MIS signaling proteins by Western blotting in basal conditions and after 6 h of incubation with LRMIS (1.6-25 nM). In all four cell lines (data not shown), basal expression of ALK2 was decreased upon incubation with LRMIS and was nearly undetectable in the presence of 6.25 or 12.5 nM LRMIS. Conversely, ALK3 expression was increased upon exposure to LRMIS. Furthermore, SMAD1 / 5 phosphorylation, caspase-3 / 7 activity, and caspase-3 and PARP cleavage were increased in parallel with ALK3 expression (data not shown).

[0157] To analyze the involvement of non-SMAD pathways in MIS signaling ( Beck et al., 2016 ; Zhang, 2017 ), we monitored AKT phosphorylation and found that its phosphorylation was reduced upon incubation with LRMIS, as observed for ALK2 expression (data not shown).

[0158] These results confirmed that ALK3 is the major MISRI in MIS signaling through the SMAD pathway to induce apoptosis (starting at around 6 nM LRMIS) in ovarian carcinoma cells. ALK2 is expressed in basal conditions (around 10 pM MIS), and its expression is then reduced upon incubation with LRMIS.

[0159] Anti-MIS antibody B10 reduces cell proliferation and induces growth inhibition in ovarian cancer cells To investigate whether the proliferative effect of low concentrations of MIS can be blocked by antibodies as a promising therapeutic strategy, we generated a new MAb against MIS. The B10 antibody was isolated from the human scFv phage display library Husc I (Philibert et al., 2007; Robin and Martineau, 2012) after panning with the Ala453-Arg560 MIS cTER domain, which is bioactive despite being less active than truncated MIS (Nachtigal and Ingraham, 1996; Wilson et al., 1993). First, we measured the affinity (EC 50 =50.4±1.2 nM) and its ability to inhibit the apoptotic effect of 25 nM LRMIS in COV434-MISRII and SKOV3-MISRII cells (data not shown). Caspase-3 / 7 activity induced by 25 nM MIS (fold change compared to untreated cells) was reduced by about 40% in the presence of about 66 nM B10.

[0160] We then evaluated the effect of B10 on cell viability in the presence of low LRMIS concentrations (0.1-0.6 nM). Depending on the cell line, B10 induced a decrease in cell viability of 25% (OVCAR8) to 50% (KGN) at concentrations ranging from 3 to 333 nM (data not shown). Furthermore, 333 nM B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-MISRII, SKOV3-MISRII, OVCAR8 and KGN cells, respectively (Figure 4). In the four cell lines, B10 reduced AKT phosphorylation and increased PARP and caspase 3 cleavage (data not shown), a phenomenon that was only observed early at high LRMIS concentrations (Figures 2A and B).

[0161] Finally, we evaluated the effect of B10 in primary cancer cells isolated from ascites samples of two ovarian cancer patients. These patients were waiting for surgical intervention and had never received chemotherapy. Similar to the four cell lines, B10 reduced cell viability by 30% and 20% (patients 1 and 2, respectively) (data not shown) and inhibited cell growth (estimated by confluency area) by 25% and 65% (patients 1 and 2, respectively) (Figure 5A), while B10 increased caspase-3 / 7 activity up to 3-fold (Figure 5B). Despite the limited number of samples, these results highlight the prospect of a promising discovery medicine to block MIS proliferation effects with specific antibodies.

[0162] Anti-AMH antibody B10 reduces COV434-MISRII tumor growth in vivo To evaluate whether the in vitro antiproliferative effects of B10 could be translated into in vivo antitumor activity, we treated mice (5-7 mice / group) bearing established COV434-MISRII cell-derived tumors with B10 (anti-AMH antibody), 12G4 (anti-AMHRII antibody) (both at 10 mg / kg / injection) or vehicle (NaCl) by ip injection twice weekly for 4 weeks. Both B10 and 12G4 inhibited tumor growth compared to vehicle (p<0.001) (Figure 6A). 50% of mice grew tumors up to 1,500 mm 3Median survival, defined as the time to tumor bearing, was 60, 69, and 76 days for mice treated with vehicle, B10, and 12G4, respectively (12G4 and B10, p=0.0050 and p=0.0173 vs. control; p=0.4331 between 12G4 and B10) (Figure 6B).

[0163] Consideration Here, using two ovarian cancer cell lines (COV434-MISRII and SKOV3-MISRII), we found that ALK3 is the preferred MISRI for MIS signaling and apoptosis induction.In four ovarian cancer cell lines (COV434-MISRII, SKOV3-MISRII, OVCAR8 and KGN), we showed that ALK2 and ALK3 were modulated by incubation with LRMIS, and that ALK3 was preferentially expressed and induced apoptosis when using high doses of LRMIS (Figures 2A and 2B).These results, confirmed in tumor cells isolated from ascites samples of two ovarian malignant tumor patients, are currently being used to develop new therapeutic strategies.

[0164] Based on the observation by RE Scully that epithelial ovarian malignant tumors histologically resemble tissue derived from the Müllerian duct (Scully, 1970), MIS has been proposed as a promising treatment for gynecological tumors since 1979 (Donahoe et al., 1979). Many studies reviewed by Kim JH et al. have verified the potential application of MIS as a biopharmaceutical for cancer treatment in ovarian cancer (Anttonen et al., 2011; Fuller et al., 1982; Masiakos et al., 1999; Pieretti-Vanmarcke et al., 2006; Stephen et al., 2002), cervical and endometrial cancer (Barbie et al., 2003; Renaud et al., 2005), as well as non-Müllerian tumors such as breast cancer (Gupta et al., 2005) and prostate cancer (Hoshiya et al., 2003) (Kim et al., 2014). Specifically, these studies showed that high doses of MIS can inhibit cancer cell growth in vitro and in vivo in cell lines and patient samples. Interestingly, recent results suggested that MIS may also be effective in chemotherapy-resistant cancer cells and cancer stem cells (Meirelles et al., 2012; Wei et al., 2010). A major challenge for the clinical application of this strategy is the availability of large amounts of clinical-grade MIS. To the best of our knowledge, the state-of-the-art strategy is the one developed by Pepin et al. (i.e., LRMIS with albumin leader sequence and cleavage site modification resulting in high yields of bioactive MIS) (Pepin et al., 2013).

[0165] A commonality between these studies is that they all use high doses of MIS, typically 25-200 nM, to treat cancer cells. This concentration must be compared to the highest MIS serum concentration observed physiologically (in boys from birth to puberty), which is lower than 1 nM (around 50 ng / ml). This is entirely logical, since the strategy is based on MIS induction of apoptosis during Müllerian regression. We obtained similar results in the present study, but also noted the observation that MIS promoted cell survival / proliferation at lower concentrations (0.8 nM-6.1 nM depending on the cell line).

[0166] Moreover, Beck TN et al. showed that MIS / MISRII signaling regulates epithelial-mesenchymal transition (EMT) and promotes cell survival / proliferation in lung cancer (Beck et al., 2016). They suggested that the role of MIS / MISRII signaling in EMT regulation is important for chemotherapy resistance. In this study, we showed that the new anti-MIS MAb B10 can reduce cell viability, clonogenic survival and ATK phosphorylation in all four ovarian cancer cell lines and in tumor cells isolated from ovarian cancer ascites samples (Figure 4). All these in vitro data indicate that inhibition of physiological concentrations of AMH can mimic the effects of supraphysiological concentrations of exogenous AMH. As a first step towards in vivo proof of this concept, we showed that B10 anti-AMH antibody reduced the growth of COV434-AMHRII cell-derived tumors and significantly increased their median survival compared to the control group (without treatment) in mice (Figure 6).

[0167] Based on these results, we propose that, rather than administering high MIS doses to induce apoptosis, anti-MIS MAbs such as B10 could be an innovative therapeutic approach to suppress the proliferative effects of MIS. This strategy could be evaluated first in gynecological tumors, where the MIS / MISRII signaling pathway has been well described, and then in (i) colorectal cancer, where the MIS gene is upregulated (Pellatt et al., 2018), and (ii) colorectal cancer, where high MIS RNA expression is a poor prognostic factor (n=597 patients followed up for more than 12 years) (Uhlen et al., 2017).

[0168] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure. [Table 2] TIFF0007657785000003.tif241165 TIFF0007657785000004.tif250165 TIFF0007657785000005.tif242165 TIFF0007657785000006.tif250165 TIFF0007657785000007.tif177165

Claims

1. 1. An isolated anti-Mullerian inhibitory substance (MIS) antibody, comprising: (a) a heavy chain, wherein the variable domain comprises an H-CDR1 having the sequence set forth as SEQ ID NO:1; an H-CDR2 having the sequence set forth as SEQ ID NO:2; an H-CDR3 having the sequence set forth as SEQ ID NO:4; and (b) a light chain, wherein the variable domain comprises an L-CDR1 having the sequence shown as SEQ ID NO:5; an L-CDR2 having the sequence shown as SEQ ID NO:6; an L-CDR3 having the sequence shown as SEQ ID NO:

7.

2. 2. The isolated anti-MIS antibody of claim 1, comprising a heavy chain having the sequence shown as SEQ ID NO:8; and a light chain having the sequence shown as SEQ ID NO:

10.

3. A nucleic acid molecule encoding the anti-MIS antibody of claim 1.

4. A vector comprising the nucleic acid of claim 3.

5. A host cell transfected, infected or transformed with a nucleic acid according to claim 3 and / or a vector according to claim 4.

6. A pharmaceutical composition comprising the anti-MIS antibody of claim 1.

7. 2. The antibody of claim 1 for use in treating a MIS or MISRII positive cancer in a subject in need thereof.

8. 8. The antibody for use according to claim 7, used in combination with conventional treatment of MIS or MISRII positive cancer.

9. 9. The antibody for use according to claim 7 or 8, wherein the MIS or MISRII positive cancer is selected in the group consisting of gynecological cancer, lung cancer or colorectal cancer.

10. A pharmaceutical composition for treating gynecological cancer, lung cancer or colorectal cancer in a subject, comprising a therapeutically effective amount of an anti-MIS antibody according to claim 1.

11. A pharmaceutical composition for treating MIS or MISRII positive cancer in a subject, comprising a therapeutically effective amount of an anti-MIS antibody described in claim 1.

12. The pharmaceutical composition of claim 11, used in combination with conventional treatment for MIS or MISRII positive cancer.

13. Use of an anti-MIS antibody described in claim 1 in the manufacture of a medicament for treating gynecological cancer, lung cancer or colorectal cancer in a subject.

14. Use of an anti-MIS antibody described in claim 1 in the manufacture of a medicament for treating MIS or MISRII positive cancer in a subject.

15. The use of claim 14, wherein the medicament is used in combination with conventional treatment for MIS or MISRII positive cancer.

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