EP4669350A1 - METHOD FOR THE TREATMENT OF ENDOMETRIOSIS - Google Patents

Abstract

Endometriosis is a chronic oestrogen-dependent inflammatory disease characterised by the presence of endometrial tissue outside the uterine cavity. There is currently no cure for endometriosis. Here, the Inventors studied the impact of POM-1, an inhibitor of CD39 enzyme activity, on the endometrial microenvironment at the molecular and cellular level. Using a mouse model of endometriosis, the Inventors demonstrated in vivo that targeting CD39 modulated the endometrial microenvironment and that these modifications altered the polarisation of immune cells present in the peritoneal cavity towards a pro-inflammatory phenotype. These initial results thus demonstrate the relevance of CD39 as a therapeutic target in endometriosis.

Description

METHODS FOR THE TREATMENT OF ENDOMETRIOSIS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular gynaecology.

BACKGROUND OF THE INVENTION:

Endometriosis is a chronic oestrogen-dependent inflammatory disease characterised by the presence of endometrial tissue outside the uterine cavity. Endometriosis affects about 10% of women and girls of childbearing age worldwide, representing about 190 million people. This chronic disease is associated with painful periods, chronic pelvic pain, pain during and/or after intercourse, pain during bowel movement, pain during urination, fatigue, depression, anxiety, bloating, nausea and/or infertility. Endometriosis can be detected by clinical and ultrasound examination or even by MRI, although the definitive diagnosis is preferably made by analysis of endometrial tissue removed during surgery.

There is currently no cure for endometriosis. When a patient suffers from endometriosis, first-line hormonal treatment is offered to suppress menstruation (e.g. progestins, continuous monophasic oestrogen contraceptives, danazol or GnRH analogues), but a total elimination of the lesions is rarely achieved. Surgery is the only treatment that can completely eradicate the lesions associated with endometriosis. Surgery is performed in cases of disabling symptoms and/or infertility. With surgery, the painful symptoms can disappear for several years, or even completely. However, surgery is not effective in the case of small disseminated lesions and sometimes surgery cannot be performed because of an unfavourable benefit/risk ratio, with for example a risk of incontinence.

To date, there is still a need to complete the therapeutic arsenal to treat patients suffering from endometriosis.

SUMMARY OF THE INVENTION:

The invention is defined by the claims. In particular, the present invention relates to a method of treating endometriosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CD39 inhibitor. DETAILED DESCRIPTION OF THE INVENTION:

The Inventors studied the impact of POM- 1, an inhibitor of CD39 enzyme activity, on the endometrial microenvironment at the molecular and cellular level. Using a mouse model of endometriosis, the Inventors demonstrated in vivo that targeting CD39 modulated the endometrial microenvironment and that these modifications altered the polarisation of immune cells present in the peritoneal cavity towards a pro-inflammatory phenotype. These results thus demonstrate the relevance of CD39 as a therapeutic target in endometriosis.

Accordingly, the present invention relates to a method of treating endometriosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CD39 inhibitor.

As used herein, the term “endometriosis” refers to a disease characterized by the abnormal presence of uterine tissue (or endometrial tissue) outside the uterine cavity. The term may also encompass eutopic endometriosis and endometriotic lesions in uterine cavity. The term includes peritoneal endometriosis, ovarian endometriosis, deep endometriosis, extrapelvic endometriosis and adenomyosis. The lesions are made up of cells that have the same characteristics as those of the uterine lining (the endometrium) and behave like them under the influence of ovarian hormones. It causes chronic inflammatory reactions and lead to the formation of scar in the pelvis and other parts of the body. Typically, the most affected organs are ovaries, uterosacral ligaments, rectum, bladder and vagina. Symptoms caused by endometriosis include painful periods, chronic pelvic pain, pain during and/or after intercourse, pain during bowel movement, pain during urination, fatigue, depression, anxiety, bloating and/or nausea. In addition, endometriosis can lead to infertility. Accordingly in some embodiments, the method of the invention is particularly suitable to alleviate at least one symptom caused by endometriosis selected from the list comprising painful periods, chronic pelvic pain, pain during and/or after intercourse, pain during bowel movement, pain during urination, fatigue, depression, anxiety, bloating and/or nausea. As example, to be diagnosed, endometriomas (i.e. clumps of tissue) are sought through ultrasound or magnetic resonance imaging (MRI) techniques. Histological examinations can be used to confirm a diagnosis (e.g. during a surgery).

As used herein, the term “subject” or “patient” denotes a mammal, preferably female. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with endometriosis. In some embodiments, the subject has undergone or will undergo endometriosis surgery.

As used herein, the term “endometriosis surgery” refers to a surgery aiming to remove or destroy the deposits of endometriosis. The term encompasses surgery to cut away patches of endometriosis tissue, surgery to remove part or all of the organs affected by endometriosis (e.g. hysterectomy, oophorectomy) or both. As example, an endometriosis surgery can be performed with laparoscopy or laparotomy.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). More particularly, the term “treatment of endometriosis” includes treatment to reduce or remove the amount of endometrial tissue which is present inside and/or outside the uterine cavity (e.g. reduction or removal of endometriotic lesions); and/or treatment to reduce and/or ameliorate one or more symptoms associated with endometriosis. The American Society for Reproductive Medicine (ASRM) defines a classification system for the various stages of endometriosis, dividing this into four stages (stage IV most severe; stage I least severe) [American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997; 67,817 821.]. Thus, the term “treatment of endometriosis” includes treatment to reduce the severity of the condition as measured by ASRM classification, e.g. treatment to reduce the severity of the endometriosis from Stage IV, III, II, or I to a lower stage, or until the symptoms are completely alleviated.

As used herein, the term “CD39” has its general meaning in the art and refers to the CD39 protein, also named as EctoNucleoside TriphosPhate Diphosphohydrolase- 1 (ENTPD1). CD39 is an ectoenzyme that hydrolyzes ATP/UTP and ADP/UDP to AMP. CD39 is encoded by ENTPD1 gene (Entrez Gene: 953; Ensembl: ENSG00000138185). An exemplary amino acid sequence for CD39 is represented in SEQ ID NO:1.

SEQ ID NO : 1 >sp | P49961 | ENTP1_HUMAN OS=Homo sapiens OX=9606 GN=ENTPD1 PE=1 SV=1 MEDTKESNVK TFCSKNILAI LGFSSI IAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT SLYIYKWPAE KENDTGWHQ VEECRVKGPG I SKFVQKVNE IGIYLTDCME RAREVI PRSQ HQETPVYLGA TAGMRLLRME SEELADRVLD WERSLSNYP FDFQGARI IT GQEEGAYGWI TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR LYGKDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGI FL PPLQGDFGAF SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EYCFSGTYIL SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MI PAEQPLST PLSHSTYVFL MVLFSLVLFT VAI IGLLI FH KPSYFWKDMV

As used herein, the term “CD39 inhibitor” refers to a molecule that partially or fully blocks, inhibits, or neutralizes a biological activity or expression of CD39. A CD39 inhibitor can be a molecule of any type that interferes with the signalling associated with CD39 in a cell, for example, either by decreasing transcription or translation of CD39-encoding nucleic acid, or by inhibiting or blocking CD39 polypeptide activity, or both. Examples of CD39 inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, CD39-specific aptamers, anti-CD39 antibodies, CD39-binding fragments of anti-CD39 antibodies, CD39-binding small molecules, CD39-binding peptides, and other polypeptides that specifically bind CD39 (including, but not limited to, CD39-binding fragments of one or more CD39 ligands, optionally fused to one or more additional domains), such that the interaction between the CD39 inhibitor and CD39 results in a reduction or cessation of CD39 activity or expression.

In some embodiments, the CD39 inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not). The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In some embodiments, the CD39 inhibitor is POM-1. As used herein, the term “POM- 1” refers to an inhibitor of Ecto-NTPDases (CAS No: 12141-67-2).

In some embodiments, the CD39 inhibitor is an antibody having specificity for CD39. In some embodiments, the CD39 inhibitor is an anti-CD39 antibody. As used herein, the term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc- diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody -based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.

The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H- - 9 - CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. (http://www.bioinf.org.Uk/abs/#cdrdef)

In some embodiments, the amino acid residues of the antibody of the invention are numbered according to the IMGT numbering system. The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the 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 15 for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains" Dev. Comp. Immunol., 27, 55- 77 (2003).). In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cysteine 23, tryptophan 41, hydrophobic amino acid 89, cysteine 104, phenylalanine or tryptophan 118. The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1- 20 IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the top of the loop, in the following order 111, 112, 110, 113, 109, 114, etc. If the CDR3-IMGT length is more 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-FRCDRdefmition.html).

As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as CD39, while having relatively little detectable reactivity with non-CD39 proteins or structures (such as other proteins presented on endometriosis lesions). Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is a CD39 polypeptide).

The term “affinity”, as used herein, means the strength of the binding 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 the unbound antibody and [Ag] is the molar concentration of the 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, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

The term “Fab” denotes an antibody fragment having a molecular weight of about 50,000Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond. The term “F(ab')2” refers to an antibody fragment having a molecular weight of about 100,000Da and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin. The term “Fab'”refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2. A single chain Fv (“scFv”) polypeptide is a covalently linked VH: : VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. “dsFv” is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

The term "diabodies" refers to small antibody fragments with two antigen -binding sites, which fragments comprise 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 the complementary domains of another chain and create two antigen-binding sites.

Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with the appropriate antigenic forms (i.e. CD39 or cell that express CD39). Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods. Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as nondenaturing ELISA, flow cytometry, and immunoprecipitation. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated as Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

In some embodiments, the antibody is a humanized antibody. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.

In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans. In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

The antibody of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions, such as ADCC induction. Exemplary isotypes are IgGl, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a human monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the human monoclonal antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In some embodiments, the antibody of the present invention is a full- length antibody. In some embodiments, the full-length antibody is an IgGl antibody. In some embodiments, the full-length antibody is an IgG4 antibody. In some embodiments, the specific IgG4 antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibodies are antibodies wherein arginine at position 409 in a heavy chain constant region of human IgG4, which is indicated in the EU index as in Kabat et al. supra, is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in W02006/033386) and/or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence. Other suitable stabilized IgG4 antibodies are disclosed in WO2008/145142, which is hereby incorporated by reference in its entirety. In some embodiments, the human monoclonal antibody of the present invention is an antibody of a non-IgG4 type, e.g. IgGl, IgG2 or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).

Monoclonal antibodies that are CD39 inhibitors are well known in the art and includes as example those described in the international patent applications W02009/095478, WO2012/085132, WO2016/073845, WO2021/055329, WO2021/056610, WO2022/111576. As example, antibodies having specificity for CD39 includes, but are not limited to, TTX-030 (Trishula Therapeutics), TTX-030-001 (Trishula Therapeutics), TTX-030-002 (Trishula Therapeutics), IPH52 (Innate Pharma), IPH5201 (Innate Pharma), OREG-103/BY40 (Orega Biotech), BA54g (Orega Biotech), BY12 (Orega Biotech), SRF370 (Surface Oncology), SRF365 (Surface Oncology), SRF367 (Surface Oncology), SRF617 (Perenostobart, Surface Oncology), 9-8B (Igenica Therapeutics), ES014 (Elpiscience Biopharma), ES002 (Eurestobart, Elpiscience Biopharma), ES002023 (Elpiscience Biopharma), EMB04 (EpimAb Biotherapeutics) or AB598 (Arcus Biosciences). In some embodiments, the antibody having specificity for CD39 is selected from the list comprising TTX-030, IPH5201, BY40, BA54g, BY12, SRF617, 9-8B, ES002023 or ES014.

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:2. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:3 (SYEMH), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:4 (RINPSVGSTWYAQKFQG) and the VH-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:5 (GKREGGTEYLRK).

SEQ ID NO : 2 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 )

QVQ L VQ S GAE VKK P GAS VKVS C KAS G YT FKSYEMHWVRQAP GQGLEWMGRINPSVGSTWYAQKFQGRVT MT RDT S T S T VYMEL S S LRS EDTAVYYCARGKREGGTEYLRKWGQGT LVT VS S

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO:6. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:7 (RASQSVASSYLA), the VL-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:8 (GASNRHT) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:9 (QQYHNAIT).

SEQ ID NO : 6 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) EIVLTQSPGTLSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGI PDRFSGSGSG TD FT LT I S RLEPEDFAVYYCQQYHNAITFGGGT KVE I K

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:10. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:11 (DYNMH), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO: 12 (YIVPLNGGSTFNQKFKG) and the VH-CDR3 of the anti- CD39 antibody is defined by SEQ ID NO:13 (GGTRFAY).

SEQ ID NO : 10 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGRTLEWIGYIVPLNGGSTFNQKFKGRA TLTVNT S S RTAYMELRS LT S EDSAAYYCARGGTRFAYWGQGTLVTVSA

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO: 14. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO: 15 (RASESVDNFGVSFMY), the VL-CDR2 of the anti- CD39 antibody is defined by SEQ ID NO: 16 (GASNQGS) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO: 17 (QQTKEVPYT). SEQ ID NO : 14 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2-

FR3-CDR3- FR4 )

DIVLTQSPASLAVSLGQRATI SCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGASNQGSGVPARFRGS

GS GT D FS LN I H PMEADDTAMYFCQQTKEVPYT FGGGT KLE I K

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:18. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO: 19 (GTFSSEGIS), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NQ:20 (SILPIFGTANYAQKFQG) and the VH-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:21 (AREAGYYRYRYFDL).

SEQ ID NO : 18 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 )

QVQ L VQ S GAE VKK P G S S VKVS C KAS GGTFSSEGI SWVRQAP GQGLEWMGS FGTANYAQKFQGRVT ITADESTSTAYMELSSLRSEDTAVYYCAREAGYYRYRYFDLWGKGTLVTVSS

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO:22. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:23 (RASQSVSSNLA), the VL-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:24 (GASTRAT) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:25 (QQHALWPLT).

SEQ ID NO : 22 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 )

E I VMT Q S P AT L S VS P GE RAT L S C RASQSVSSNLAW YQQ K P GQAP RL L I YGASTRAT G I P ARE S G S G S GT EFT LT I S S LQ S ED FAVYYCQQHALWPLT FGGGT KVE I K

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:26. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:27 (GYTFTHYG), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:28 (INTYTGEP) and the VH-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:29 (ARRRYEGNYVFYYFDYWGQGTTLTVSS).

SEQ ID NO : 26 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) TRVKKPRETVKI SCKASGYTFTHYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLEASVS TAYLQINNLKNEDTATYFCARRRYEGNYVFYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMV TLGCLVKGYFPEQVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPS

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO:30. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:31 (RASENIYSYFS), the VL-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:32 (TAKTLAE) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:33 (QHHYVTPYTFGGGTKLEIKR).

SEQ ID NO : 30 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 )

DI QMTGS PAS LSAS VGETVT I TCRASENI YSYFSWYQQKQGKS P Q L L VYT AKT LAE GVP S RES GS GS GT QFSLKINSLQPEDFGSYYCQHHYVTPYTFGGGTKLEIKRADAAPTVSI FPPSSEQLTSGGASWCFLNN FYPKDINVKWKIDGSERQNGVLNSWTD

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:34. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:35 (GGSRKLSCAASGFTFSSFGMH), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:36 (YISSGSSIIYYADTVKG) and the VH- CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:37 (WSTTVVATDYWGQGTTLTVS).

SEQ ID NO : 34 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYI SSGSSI IYYADTVKGRFT I S RDN P KNT L FLQMT S LGS EDTAMYYCARWSTTWATDYWGQGTTLTVS

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO:38. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:39 (KASENVVTYVS), the VL-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:40 (GASNRYT) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:41 (CGQGYSYPYTFGGGTKLEIKR).

SEQ ID NO : 38 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) NIVMTQSPKSMSMSVGERVTLTCKASENWTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSAT

DFTLTI S SVQAEDLADYHCGQGYSYPYTFGGGTKLEIKR

In some embodiments, the VH domain of the anti-CD39 antibody consists in the sequence of SEQ ID NO:42. According to this embodiment, the VH-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:43 (HYGMN), the VH-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:44 (WINTYTGELTYADDFKG) and the VH-CDR3 of the anti- CD39 antibody is defined by SEQ ID NO:45 (RAYYRYDYVMDY).

SEQ ID NO : 42 > VH domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 )

QI QLVQS GPELKKPGETVKI S CKAS GYT FTHYGMNWVKQAPGKGLKWMGWINTYTGELTYADDFKGRFA FSLETSASTAYLQINNLKNEDTATYFCARRAYYRYDYVMDYWGQGTSVTVSS

In some embodiments, the VL domain of the anti-CD39 antibody consists of the sequence of SEQ ID NO:46. According to this embodiment, the VL-CDR1 of the anti-CD39 antibody is defined by SEQ ID NO:47 (KASHNVGTNVA), the VL-CDR2 of the anti-CD39 antibody is defined by SEQ ID NO:48 (SASYRYS) and the VL-CDR3 of the anti-CD39 antibody is defined by SEQ ID NO:49 (HQYNNYPYT).

SEQ ID NO : 46 > VL domain of the anti-CD39 antibody ( FR1-CDR1-FR2-CDR2- FR3-CDR3- FR4 ) DIVMTQSQKFMSTSVGDRVSVTCKASHNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPGRFTGSGSGT DFTLTI SNVQSEDLAEYFCHQYNNYPYTFGGGTKLEIK

In some embodiments, the anti-CD39 antibody comprises a VH domain selected from the group consisting in SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:34 or SEQ ID NO:42, and a VL domain selected from the group consisting in SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38 or SEQ ID NO:46. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:2 and a VL domain that consists of the sequence as set forth in SEQ ID NO:6. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 10 and a VL domain that consists of the sequence as set forth in SEQ ID NO: 14. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 18 and a VL domain that consists of the sequence as set forth in SEQ ID NO:22. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:26 and a VL domain that consists of the sequence as set forth in SEQ ID NO:30. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO:34 and a VL domain that consists of the sequence as set forth in SEQ ID NO:38. In some embodiments, the anti-CD39 antibody comprises a VH domain that consists of the sequence as set forth in SEQ ID NO: 42 and a VL domain that consists of the sequence as set forth in SEQ ID NO:46.

In some embodiments, the antibody of the present invention is a single chain antibody. As used herein the term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06/030220, WO 06/003388. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FR1 as "Framework region 2" or "FR2"; as "Framework region 3 " or "FR3"; and as "Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

In some embodiments, the antibody leads to the depletion of CD39 expressing endometriosis cells. As used herein, the term “depletion” with respect to endometriosis cells, refers to a measurable decrease in the number of CD39 expressing endometriosis cells in the patient. The reduction can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the term refers to a decrease in the number of CD39 endometriosis cells in the patient below detectable limits. In some embodiments, the antibody suitable for depletion of CD39 endometriosis cells mediates antibody-dependent cell-mediated cytotoxicity. As used herein the term “antibodydependent cell-mediated cytotoxicity” or “ADCC” refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., T gamma delta lymphocytes, Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).

In some embodiments, the CD39 inhibitor is an inhibitor of CD39 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, antisense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of CD39 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of CD39, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding CD39 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression in the method of the present invention. CD39 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CD39 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing CD39. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

By a "therapeutically effective amount" of the inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. Typically, the inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially 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. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable 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 form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the inhibitor at the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Any mode of administration that produces desired therapeutic effect without unacceptable adverse effects is relevant in practicing the invention. Such modes of administration may include oral, rectal, topical, transdermal, sublingual, intramuscular, parenteral, intravenous, intracavity, vaginal and adhesive matrix to be used during surgery. Certain carriers that may contain the CD39 inhibitor or the pharmaceutical composition comprising the CD39 inhibitor should be considered such as pill, patch, spray, injection or implant. Vaginal administration of the CD39 inhibitor is also possible, for example by vaginal pessary, vaginal tablet, vaginal ring or intrauterine systems.

In some embodiments, the CD39 inhibitor of the present invention is administered to the subject in combination with at least one conventional treatment used for the management of endometriosis and associated symptoms.

As used herein, the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third. . . ) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the patient to which the drugs are delivered.

Conventional treatment used for the management of endometriosis and associated symptoms includes but are not limited to hormone therapy (e.g. continuous monophasic estrogen-progestin contraceptives, progestins, danazol or GnRH analogues), analgesics (e.g. as paracetamol, aspirin, ibuprofen) or treatment to improve fertility (e.g. clomiphene citrate). In some embodiments, the CD39 inhibitor is administered to the subject in combination with at least one selected from the list consisting of hormone therapy, analgesics or treatment to improve fertility.

In some embodiments, the CD39 inhibitor is administered to the subject in combination with hormone therapy. Hormone therapy includes but are not limited to continuous monophasic estrogen-progestin contraceptives, progestins, danazol or GnRH analogues. In some embodiments, the hormone therapy is selected from chlormadinone acetate, cyproterone acetate, danazol, desogestrel, drospirenone, dienogest, 5a-dihydroprogesterone, ethanediol acetate, ethynodiol diacetate, etonogestrel, gestodene, estretol, 17-hydroxyprogesterone, levomefolate, levonorgestrel, medroxyprogesterone acetate (17a-hydroxy-6a- methylprogesterone acetate), megestrol, megestrol acetate (17aacetoxy-6-dehydro-6- methylprogesterone), nestorone, nomegestrol acetate, norethindrone, norethindrone acetate, norethynodrel, norgestimate, norgestrel, progesterone, tanaproget, trimegestone, danazol, leuprolide, leuprolide acetate, goserelin, goserelin acetate, histrelin, histrelin acetate, triptorelin, nafarelin, degarelix, elagolix, pharmaceutically acceptable salts of any of the foregoing, and any combination thereof.

In some embodiments, the CD39 inhibitor is administered to the subject in combination with analgesics. Analgesics includes but are not limited to paracetamol, aspirin, non-steroidal anti-inflammatory drugs, codeine, morphine, tramadol, corticosteroids, antispasmodics, local anaesthetics, general anaesthetics.

Thus, in some embodiments, the CD39 inhibitor is administered to the subject in combination with at least one selected from the list comprising or consisting in chlormadinone acetate, cyproterone acetate, danazol, desogestrel, drospirenone, dienogest, 5a- dihydroprogesterone, ethanediol acetate, ethynodiol diacetate, etonogestrel, gestodene, estretol, 17-hydroxyprogesterone, levomefolate, levonorgestrel, medroxyprogesterone acetate (17a-hydroxy-6a-methylprogesterone acetate), megestrol, megestrol acetate (17aacetoxy-

6-dehydro-6-methylprogesterone), nestorone, nomegestrol acetate, norethindrone, norethindrone acetate, norethynodrel, norgestimate, norgestrel, progesterone, tanaproget, trimegestone, danazol, leuprolide, leuprolide acetate, goserelin, goserelin acetate, histrelin, histrelin acetate, triptorelin, nafarelin, degarelix, elagolix, paracetamol, aspirin, non-steroidal anti-inflammatory drugs, codeine, morphine, tramadol, corticosteroids, antispasmodics, local anaesthetics, general anaesthetics, clomiphene citrate, pharmaceutically acceptable salts of any of the foregoing, and any combination thereof.

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

FIGURES:

Figure 1. Experimental scheme of the endometriosis mouse model and the implementation of POM-1 administration

Figure 2. Macrophages analysis at D7

Figure 3. CD4+ T cells polarisation analysis at D7

Figure 4. Anti-CD39 Ab administration

Figure 5. Anti-CD39 Ab administration

EXAMPLE 1:

Material and Methods

Donor Mice. 42 hours before the endometriosis induction operation, PMSG (Pregnant Mare Serum Gonadotrophin; 20IU/100pl) was injected intraperitoneally in order to induce endometrial decidualization. On the day of surgery, these mice were killed by cervical dislocation and their uteri were recovered. Endometrium preparation. Each uterine horn was cut longitudinally with a scalpel blade and then dilacerated with a biopsy punch (3mm) in a petri dish containing IX PBS (Phosphate-Buffered Saline). Then, the tissue pieces were evenly distributed in 1ml syringes in 300pl of PBS and 400pl of air was added to each syringe before injection.

Recipient mice. Mice were anesthetized by intraperitoneal injection of a mixture of Ketamine (150 mg/kg) and Xylazine (10 mg/kg). Once the mice were anesthetized, the right flank and peritoneum were incised over a 4-mm-wide area and the uterine horn pieces contained in the syringe were injected into the peritoneal cavity. Each of the recipient mice received the equivalent of one uterine horn. The peritoneum of the operated mice was then reattached with surgical glue and the skin was stapled.

Estrogen injection. In order to mimic the development of endometriosis, a subcutaneous injection of estrogen diluted in sesame oil (2.5 pg Beta estradiol/lOOpl/mouse) was performed after the operation and then every 7 days during the follow-up of the mice. At the end of the experimental protocol, the mice were killed by cervical dislocation at different postoperative times and the samples to be analyzed were recovered.

CD39 inhibitor (POM-1) administration. Recipient mice were randomized into two groups of equal size. Untreated mice received lOOpl per day of PBS, a saline solution serving as a control, while treated mice received POM-1, a pharmacological inhibitor of CD39, (10 mg/kg/day in ~100pl) according to the treatment protocol described by S. Robson's team.

Results

To define the impact of inhibition of CD39 enzyme activity, mice were treated daily with the inhibitor (POM-1) from implantation until sacrifice (D3, D7 or D14). At necropsy, we observed that the size of the lesions was significantly smaller in the treated mice than in the untreated ones. These lesions showed less angiogenesis and adhesions with neighboring tissues. All the recovered lesions were then weighed, and we observed a significant decrease in the weight of the lesions in the treated mice (Figure 1). These first results show a beneficial effect of the treatment on the development of endometriosis.

We analyzed the polarization of macrophages present in the peritoneal fluids of treated and untreated mice at D7 after surgery. Mice were sacrificed and the polarization of myeloid cells in the peritoneal cavity was defined by flow cytometry. The cells were labeled with antibodies against the surface proteins CD45 (hematopoietic cells), CDl lb (myeloid cells), F4/80 (macrophages), and CD38 (pro-inflammatory macrophages). To define the phenotype of the macrophages present, we select the viable cells that express CD45, CD1 lb, F4/80 on which we analyze the level of CD38 expression.

Analysis of macrophages polarization shows a significant increase in pro-inflammatory macrophages (CD38+) associated with a significant decrease in regulatory macrophages (CD38-) after 7 days of treatment (Figure 2).

Macrophages have a pivotal role in the polarization of the microenvironment and the activation of immune cells such as T lymphocytes. Based on the above observations, we therefore defined the polarization of T lymphocytes present in the peritoneal cavity of POM-1 treated and untreated mice. The intracellular expression of transcription factors specific to LT subpopulations: T-bet (Thl LT, CD8+ Tel LT (Tel LT)), RORyT (Th 17 LT, CD8+ Tcl7 LT (Tcl7 LT)), Foxp3 (LTreg) present in the CD1 lb- cell fraction was analyzed by flow cytometry. After selecting leukocytes (based on their size and granulosity) and excluding doublets, we analyzed the expression level of transcription factors within CD4 (CD3+CD4+) and CD8 (CD3+CD4-) LTs. At D7, we observe that treatment induces a significant decrease in Thl7 LTs (RORyt+) as well as CD4 LTs that coexpress RORyt and T-bet. We also observe a decrease in the percentage of Tcl7 (RORyt+) LTs and T-bet+ RORyt+ CD8 LTs (Figure 3).

EXAMPLE 2:

Material and Methods

Donor Mice. 42 hours before the endometriosis induction operation, PMSG (Pregnant Mare Serum Gonadotrophin; 20IU/100pl) was injected intraperitoneally in order to induce endometrial decidualization. On the day of surgery, these mice were killed by cervical dislocation and their uteri were recovered.

Endometrium preparation. Each uterine horn was cut longitudinally with a scalpel blade and then dilacerated with a biopsy punch (3mm) in a petri dish containing IX PBS (Phosphate-Buffered Saline). Then, the tissue pieces were evenly distributed in 1ml syringes in 300pl of PBS and 400pl of air was added to each syringe before injection. Recipient mice. Mice were anesthetized by intraperitoneal injection of a mixture of Ketamine (150 mg/kg) and Xylazine (10 mg/kg). Once the mice were anesthetized, the right flank and peritoneum were incised over a 4-mm-wide area and the uterine horn pieces contained in the syringe were injected into the peritoneal cavity. Each of the recipient mice received the equivalent of one uterine horn. The peritoneum of the operated mice was then reattached with surgical glue and the skin was stapled.

Estrogen injection. In order to mimic the development of endometriosis, a subcutaneous injection of estrogen diluted in sesame oil (2.5 pg Beta estradiol/lOOpl/mouse) was performed after the operation and then every 7 days during the follow-up of the mice. At the end of the experimental protocol, the mice were killed by cervical dislocation at different postoperative times and the samples to be analyzed were recovered.

Anti-CD39 antibody administration. Recipient mice were randomized into two groups of equal size. Treated mice received an anti-CD39 antibody (Ivan Perrot et al. Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies, Cell Reports, Volume 27, Issue 8, 2019, Pages 2411-2425. e9, ISSN 2211-1247), a blocking antibody of CD39 enzymatic activity at 20 mg/kg at day 0 and 10 mg/kg at day 2 and 4 in ~200pl of PBS while untreated mice received the same quantity of control antibody (20 mg/kg at day 0 and 10 mg/kg at day 2 and 4 in ~200pl of PBS).

Results

To define the impact of inhibition of CD39 on endometriotic lesions, mice were treated with an anti-CD39 antibody. We observed a decrease in the weight of the lesions in treated mice (Figure 4 and Figure 5). These results show a beneficial effect of the treatment on the development of endometriosis.

CONCLUSION

We have shown that inhibition of CD39 activity by POM-1 significantly alters the microenvironment and polarization of immune cells present in the peritoneal cavity of modeled mice and that these alterations impair the development of endometriosis. We also demonstrated in vivo that targeting CD39 with an anti-CD39 antibody alters the development of endometriosis. REFERENCES:

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

Claims

CLAIMS:

1. A method of treating endometriosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a CD39 inhibitor.

2. The method according to claim 1, wherein the endometriosis is peritoneal endometriosis, ovarian endometriosis, deep endometriosis, extrapelvic endometriosis or adenomyosis.

3. The method according to claim 1 or 2 to alleviate at least one symptom caused by endometriosis selected from the list comprising painful periods, chronic pelvic pain, pain during and/or after intercourse, pain during bowel movement, pain during urination, fatigue, depression, anxiety, bloating and/or nausea.

4. The method according to any of claims 1 to 3, wherein the CD39 inhibitor is an antibody having specificity for CD39.

5. The method according to claim 4, wherein the antibody having specificity for CD39 is selected from the list comprising TTX-030, IPH5201, BY40, BA54g, BY12, SRF617, 9-8B, ES002023 or ES014.

6. The method according to any of claims 1 to 5, wherein the CD39 inhibitor is administered to the subject in combination with at least one selected from the list consisting of hormone therapy, analgesics or treatment to improve fertility.

7. The method according to any of claims 1 to 6, wherein the CD39 inhibitor is administered to the subject in combination with at least one selected from the list comprising chlormadinone acetate, cyproterone acetate, danazol, desogestrel, drospirenone, dienogest, 5a-dihydroprogesterone, ethanediol acetate, ethynodiol diacetate, etonogestrel, gestodene, estretol, 17-hydroxyprogesterone, levomefolate, levonorgestrel, medroxyprogesterone acetate (17a-hydroxy-6a-methylprogesterone acetate), megestrol, megestrol acetate (17aacetoxy-6-dehydro-6- methylprogesterone), nestorone, nomegestrol acetate, norethindrone, norethindrone acetate, norethynodrel, norgestimate, norgestrel, progesterone, tanaproget, trimegestone, danazol, leuprolide, leuprolide acetate, goserelin, goserelin acetate, histrelin, histrelin acetate, triptorelin, nafarelin, degarelix, elagolix, paracetamol, aspirin, non-steroidal anti-inflammatory drugs, codeine, morphine, tramadol, corticosteroids, antispasmodics, local anaesthetics, general anaesthetics, clomiphene citrate, pharmaceutically acceptable salts of any of the foregoing, or any combination thereof.