Anti-Netrin-1 monoclonal antibody for the treatment of endometriosis and referred pain

Abstract

The present invention relates to the treatment of endometriosis using an anti-Netrin-1 antibody or an antigen-binding fragment thereof, and to such an antibody or fragment thereof for use in treating endometriosis. The treatment includes reduction of endometriotic lesions, particularly uterine and / or uterine horn endometriotic lesions. The treatment further includes relief of chronic pelvic pain or vaginal hyperalgesia. This may also include reduction of colorectal hyperalgesia developed in endometriosis, improvement of bladder dysfunction developed in endometriosis, reduction of increased skin sensitivity to heat and / or mechanical stimuli developed in endometriosis, and improvement of overall health status altered by endometriosis.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical and method for the treatment of endometriosis that enables the reduction or elimination of endometriotic lesions, and more particularly, combines favorable therapeutic effects against endometriosis and its characteristic lesions, as well as referred pain including vaginal hyperalgesia, and other comorbidities. [Background technology]

[0002] Endometriosis is a chronic inflammatory gynecological disorder affecting approximately 10% of women of reproductive age, and it accounts for an economic burden of approximately $80 billion per year in the United States alone. Chronic pelvic pain (CPP) is the most debilitating symptom experienced by women with endometriosis. Furthermore, patients with endometriosis are usually diagnosed with co-occurring visceral conditions, including irritable bowel syndrome (IBS) and overactive bladder syndrome (OAB). Recent research into the mechanisms underlying endometriotic pain has primarily focused on endometriotic lesions and their inflammatory environment as sources of CPP. However, surgical removal of endometriotic lesions does not reliably provide pain relief, and there remains a lack of adequate treatment.

[0003] According to the World Health Organization, endometriosis is a disease characterized by the presence of tissue outside the uterus that resembles the endometrium (the lining of the uterus). This causes a chronic inflammatory response that can lead to the formation of scar tissue (adhesion, fibrosis) in the pelvis and other parts of the body. Several lesion types have been described, such as superficial endometriosis, which is mainly found in the pelvic peritoneum; cystic ovarian endometriosis (endometrioma), which is found in the ovaries; and deep endometriosis, which is found in the rectovaginal septum, bladder, and intestines. Rarely, endometriosis can also occur outside the pelvis. Symptoms associated with endometriosis are diverse and include a combination of menstrual cramps, chronic pelvic pain, pain during and / or after sexual intercourse, bowel movements, painful urination, fatigue, depression or anxiety, abdominal distension, and nausea.

[0004] Currently, there is no known treatment for endometriosis. Treatment usually aims to control symptoms, which is clearly insufficient.

[0005] Thus, there is a need for novel therapeutic treatments that can reduce, limit, or regress lesions caused by endometriosis, preferably simultaneously reducing endometriosis-related hyperalgesia or pain, and, if applicable, also reducing other comorbidities or their symptoms. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2015 / 104360 [Patent Document 2] US10,494,427 [Patent Document 3] EP2050764A1 [Patent Document 4] EP0239400 [Patent Document 5] WO91 / 09967 [Patent Document 6] U.S. Patent No. 5,530,101 [Patent Document 7] U.S. Patent No. 5,585,089 [Patent Document 8] EP0592106 [Patent Document 9] EP0519596 [Patent Document 10] U.S. Patent No. 5,565,332 [Patent Document 11] U.S. Patent No. 4,444,887 [Patent Document 12] U.S. Patent No. 4,716,111 [Patent Document 13] U.S. Patent No. 5,545,806 [Patent Document 14] U.S. Patent No. 5,814,318 [Patent Document 15] International Patent Application No. WO98 / 46645 [Patent Document 16] International Patent Application No. WO98 / 50433 [Patent Document 17] International Patent Application No. WO98 / 24893 [Patent Document 18] International Patent Application No. WO98 / 16654 [Patent Document 19] International Patent Application No. WO96 / 34096 [Patent Document 20] International Patent Application No. WO96 / 33735 [Patent Document 21] International Patent Application No. WO91 / 10741 [Non-Patent Document]

[0007] [Non-Patent Document 1] https: / / clinicaltrials.gov / ct2 / show / NCT02977195 [Non-Patent Document 2] Harlow et al., eds., 1988 "Antibodies: a laboratory manual" [Non-Patent Document 3] Scott J.K., Smith G.P. Science 1990; 249: 386 - 390 [Non-Patent Document 4]<00001​​​​​​​​​​​​​​​​http: / / www.bioinf.org.uk / abs / #cdrdef [Non-licensed Document 9] Lefranc M.-P., "Unique database numbering system for immunogenetic analysis" Immunology Today, 18, 509 (1997) [Non-licensed Document 10] Lefranc M.-P., "Unique database numbering system for immunogenetic analysis" Immunology Today, 18, 509 (1997) [Non-licensed Document 11] Lefranc M.-P., "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains" The Immunologist, 7, pp. 132~136 (1999) [Non-licensed Document 12] 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 pages (2003) [Non-licensed Document 13] http: / / www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html [Non-licensed Document 14] Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13~22 pages [Non-licensed Document 15] Padlan (1991) Molecular Immunology 28(4 / 5): pages 489-498 [Non-Patent Document 16] Studnicka et al. (1994) Protein Engineering 7(6):805-814. [Non-Patent Document 17] Roguska et al. (1994) Proc. Natl. Acad. Sci USA 91:969-973 [Non-Patent Document 18] Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. [Non-Patent Document 19] Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) [Non-Patent Document 20] Muller, Meth. Enzymol. 92:589-601 (1983) [Overview of the project] [Problems that the invention aims to solve]

[0008] NP137 (a monoclonal antibody (mAb) against netrin-1 developed by Patrick Mehlen

[23] ) has shown anticancer efficacy in preclinical mouse models

[15] and is currently in a Phase II clinical trial for advanced cancer (https: / / clinicaltrials.gov / ct2 / show / NCT02977195). Here, for the first time, we show that NP137 has a unique and remarkable ability in vivo to reduce both endometriosis-related chronic pelvic pain by alleviating the development of endometriotic lesions and vaginal hyperalgesia. Compelling data were obtained in two distinct mouse models of endometriosis that exhibited symptoms similar to those observed in women with endometriosis (i.e., development of endometrial lesions and widespread chronic pelvic pain). [Means for solving the problem]

[0009] In one embodiment, the present invention relates to a pharmaceutical composition for use in treating endometriosis, comprising an anti-Netrin-1 antibody or its antigen-binding fragment, or an anti-Netrin-1 antibody or its antigen-binding fragment, and a pharmaceutically acceptable medium, wherein the treatment includes the reduction of endometriotic lesions, particularly uterine and / or uterine horn endometriotic lesions.

[0010] In one embodiment, the anti-Netrin-1 antibody and its antigen-binding fragment specifically bind to a Netrin-1 epitope or a binding region having an amino acid sequence, SEQ ID NO: 3 or 33.

[0011] In another embodiment, the anti-Netrin-1 antibody and its antigen-binding fragment are as defined below by a series of six CDRs disclosed, or specific VH and VL regions disclosed.

[0012] In a preferred embodiment, the anti-Netrin-1 antibody and antigen-binding fragment include a VH and VL pair of any one of the humanized monoclonal antibodies HUM01-10.

[0013] In one preferred embodiment, the anti-Netrin-1 antibody is NP137.

[0014] In a further embodiment, the treatment includes reducing both endometriotic lesions developed in the abdominal cavity (i.e., around the uterus and / or uterine horns) and alleviating chronic pelvic pain in endometriosis.

[0015] In particular, the treatment includes reducing endometriotic lesions and / or hemorrhagic lesions. Imaging allows observation, measurement, and consideration of the reduction in the total volume of lesions and / or the volume of hemorrhagic lesions. The inventors consider a reduction to be significant when the total volume of lesions is reduced by more than 20%. In one embodiment, the antibody of the present invention enables lesion reduction of more than 20%, more than 30%, more than 40%, or more than 50%. Imaging may be performed by imaging methods used for endometriosis imaging, such as transvaginal ultrasound, magnetic resonance imaging (MRI, see

[30] for more details), and computed tomography.

[0016] Furthermore, as described in Section 4, evaluation in our model by electromyographic (EMG) signal analysis of visceral motor response (VMR) induced by vaginal distension (VD) showed that treatment with NP137 resulted in improvement of vaginal hyperalgesia developed in endometriosis. In women, we use the Visual Analog Score (VAS). Treatment is generally considered to have a certain degree of effectiveness in painful endometriosis when the pain scale shows a reduction of at least 2 VAS points (out of 10). In one embodiment, the antibody of the present invention enables a reduction of at least 2, 3, or 4 VAS. See

[10] for more details on VAS.

[0017] In a further embodiment, the treatment may have the following further technical effects or therapeutic functions: - Reduction of colorectal hyperalgesia developed in endometriosis, - 75% recovery in bladder dysfunction that developed in endometriosis. - Reduction of increased skin sensitivity to heat stimuli that developed in endometriosis. - Reduction of increased skin sensitivity to mechanical stimuli that develops in endometriosis. - Improvement of overall health status altered by endometriosis It may further include one or more of the following.

[0018] In one embodiment, the present invention relates to a method for treating endometriosis in a woman in need thereof, comprising the step of administering to the woman a pharmaceutical composition containing an efficient amount of anti-Netrin-1 antibody or its antigen-binding fragment, or anti-Netrin-1 antibody or its antigen-binding fragment, and a pharmaceutically acceptable medium.

[0019] The method includes a step of reducing endometriotic lesions, particularly endometriotic lesions in the uterus and / or uterine horn.

[0020] In a further embodiment, the method includes steps of reducing endometriotic lesions, particularly endometriotic lesions in the uterus and / or uterine horn, and reducing chronic pelvic pain or vaginal hyperalgesia.

[0021] In a further embodiment, the method has the following further effects: - Reduction of colorectal hyperalgesia developed in endometriosis, - Improvement of bladder dysfunction that has developed in endometriosis, - Reduction of increased skin sensitivity to heat stimuli that developed in endometriosis. - Reduction of increased skin sensitivity to mechanical stimuli that develops in endometriosis. - Improvement of overall health status altered by endometriosis This further includes one or more of the following.

[0022] The percentages of reduction or improvement of these effects disclosed previously also apply here.

[0023] In another embodiment, the present invention relates to the use of an anti-Netrin-1 antibody or its antigen-binding fragment disclosed herein for the manufacture of a medicament for the treatment of endometriosis, preferably endometriosis and pain.

[0024] Detailed explanation The antibody may be a polyclonal or monoclonal antibody (anti-netrin-1 antibody or antibody that binds to netrin-1) that specifically binds to netrin-1, particularly human netrin-1.

[0025] Anti-Netrin-1 polyclonal antibodies are obtained, in particular, by immunizing animals, such as rabbits, mice, and the like, with a selected amino acid sequence according to methods originally known to those skilled in the art, collecting the antiserum obtained in, for example, an immunoadsorbent containing a receptor, and then depleting it.

[0026] The amino acid sequence of Netrin-1 is as shown in SEQ ID NO: 1, and Netrin-1 may be used in whole or in part to produce polyclonal or monoclonal antibodies.

[0027] Generally, monoclonal antibodies are obtained according to conventional methods of lymphocyte fusion and hybridoma culture, as described by Kohler and Milstein (Nature, 1975, 256(5517):495-497). Other methods for preparing monoclonal antibodies are also known (Harlow et al., eds., 1988, "Antibodies: a laboratory manual"). Monoclonal antibodies can be prepared by immunizing mammals (e.g., mice, rats, rabbits, or even humans, etc.) and yielding hybridomas using the lymphocyte fusion technique (Kohler and Milstein, 1975). Alternative techniques to this conventional method exist. For example, it is possible to generate monoclonal antibodies by expressing nucleic acids cloned from hybridomas. Antibodies can also be generated by phage display techniques, which involve introducing cDNA for an antibody into a vector, which is a filamentous phage exhibiting a gene library V on its surface (e.g., fUSE5 for Escherichia coli, Scott JK, Smith GP Science 1990; 249:386-390). Protocols for constructing these antibody libraries are described by JD Marks et al., J. Mol. Biol., 222 (1991), p. 581. cDNA corresponding to full-length Netrin-1 (SEQ ID NO: 2) or a preferred fragment thereof with a signal sequence may be used to generate monoclonal antibodies according to these methods.

[0028] The anti-Netrin-1 monoclonal antibody (mAb) may be a mouse, chimeric, humanized, or fully human monoclonal antibody. The fragment may be any type of mAb fragment that substantially maintains the ability of the whole antibody to bind to Netrin-1, for example, Fab or F(ab')2. In particular, the monoclonal antibodies are disclosed in WO2015 / 104360 or US10,494,427, which are incorporated herein by reference and disclose useful mouse, chimeric, and humanized monoclonal antibodies, as well as methods for preparing the various mAbs presented herein (e.g., HUM01-10 and similar). These are antibodies or fragments thereof that specifically bind to the Netrin-1 epitope, or polypeptides having an amino acid sequence, SEQ ID NO: 3 or 33, or variants thereof.

[0029] Antibodies useful in the present invention may be defined by their CDRs. In particular, these CDRs are derived from mouse antibody 4C11 disclosed in WO2015 / 104360 or US10,494,427, which are antibodies that specifically bind to polypeptides having the amino acid sequence, SEQ ID NO: 3 or 33. Preferably, the antibody is - H-CDR1 having the sequence described in Sequence ID No. 5, - H-CDR2 having the sequence described in Sequence ID No. 6, - H-CDR3 having the sequence described in Sequence ID No. 7 Variable domain VH, - L-CDR1 having the sequence described in Sequence ID No. 8, - L-CDR2 having sequence YAS, - L-CDR3 having the sequence described in Sequence ID No. 9 Variable domains (VL) including or - H-CDR1 having the sequence described in Sequence ID No. 28, - H-CDR2 having the sequence described in Sequence ID No. 29, - H-CDR3 having the sequence described in Sequence ID No. 30 Variable domain VH, - L-CDR1 having the sequence described in Sequence ID No. 31, - L-CDR2 having the sequence described in Sequence ID No. 32, - L-CDR3 having the sequence described in Sequence ID No. 9 Variable domain (VL) It is a monoclonal antibody or its antigen-binding fragment, which contains [the specified substance].

[0030] In a first series of embodiments, the antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 10, 11, 12, or 13. Typically, this comprises the VH of the sequence of SEQ ID NO: 10 and the VL of the sequence of SEQ ID NO: 11, or the heavy chain of the sequence of SEQ ID NO: 12 and the light chain of the sequence of SEQ ID NO: 13.

[0031] In a second series of embodiments, the antibody is a chimeric antibody, preferably comprising VH of the sequence of SEQ ID NO: 27 and VL of the sequence of SEQ ID NO: 19.

[0032] In a third series of embodiments, the antibody is humanized. Preferably, it includes an amino acid sequence selected from the group of SEQ ID NOs: 14-18 (VL) and / or the group of SEQ ID NOs: 20-26 (VH). Typically, the antibody is humanized and includes VH having an amino acid sequence selected from the group of SEQ ID NOs: 14-18 and VL having an amino acid sequence selected from the group of SEQ ID NOs: 20-26.

[0033] The antibody preferably comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof, the mAb or fragment specifically binding to Netrin-1. The mAb may be a mouse, chimeric, humanized, or fully human monoclonal antibody. The fragment may be any type of mAb fragment that substantially maintains the ability of the whole antibody to bind to Netrin-1, for example, Fab or F(ab')2.

[0034] The specific embodiments available herein, as disclosed in previous documents, are the antibodies listed in Table 1. The first antibody listed is a chimeric 4C11 antibody containing mouse VH and VL of the mouse 4C11 antibody. HUM00 listed in Table 1 corresponds to the transplantation of mouse 4C11 CDR into human IgG1. Ten humanized mAbs HUM01 to HUM10 are humanized mAbs derived from HUM00, possessing the same CDR but with specific modifications in the FR region of human IgG. HUM03 is also known as NP137 and is currently in clinical trials. The sequence of human IgG1 CH is derived from Genbank AEL33691.1 modified R97K. The sequence of human IgG1 CL (kappa) is derived from Genbank CAC20459.1. Other allotypes are also available. The specific binding of all these mAbs, mouse, chimeric and humanized HUM01-HUM10, Fab fragment and F(ab')2 fragment to netrin-1, and their ability to inhibit netrin-1's binding to its receptor UNC5B, is shown in US10,494,427 (Example 3).

[0035] In particular, these antibodies specifically bind to polypeptides having the amino acid sequence of SEQ ID NO: 33.

[0036] [Table 1]

[0037] Preferably, the antibody is a monoclonal antibody or its antigen-binding fragment containing a pair of VH and VL sequences selected from the following pairs: SEQ ID NOs: 27 and 19, SEQ ID NOs: 20 and 14, SEQ ID NOs: 21 and 15, SEQ ID NOs: 22 and 16, SEQ ID NOs: 23 and 17, SEQ ID NOs: 24 and 17, SEQ ID NOs: 25 and 16, SEQ ID NOs: 26 and 17, SEQ ID NOs: 22 and 17, SEQ ID NOs: 25 and 18, and SEQ ID NOs: 21 and 16. More preferably, the antibody is a monoclonal antibody or its antigen-binding fragment containing SEQ ID NOs: 22 and 16, which are a pair of VH and VL sequences.

[0038] The anti-Netrin-1 antibody may further contain the human IgG1 constant heavy chain (CH) and / or the human IgG1 constant light chain (CL), particularly the human kappa constant domain.

[0039] In one embodiment, the sequence of human IgG1 CH is derived from Genbank AEL33691.1 modified R97K. The sequence of human IgG1 CL (kappa) is derived from Genbank CAC20459.1. In one embodiment, the mAb is NP137 (AB_2811180 in the Antibody Registry) and includes sequence numbers 22 and 16 as VH and VL sequences, respectively, as well as their intrinsic IgG1 CH and CL.

[0040] The term “antigen-binding fragment” (or “antibody-binding portion”) of an antibody, as used herein, refers to one or more fragments of an antibody that maintain the ability to specifically bind to Netrin-1. The antibody fragment may include, for example, a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In one embodiment, the fragment includes the VH and VL sequences of an antibody selected from HUM00 to HUM10.

[0041] Available anti-netrin-1 antibodies include other antibodies developed against human netrin-1 or animal netrin-1, particularly monoclonal antibodies or their antigen-binding fragments, as netrin-1 is highly homologous between species. Examples include Abcam antibodies ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, and AF128.

[0042] In one embodiment, an antibody or its antigen-binding fragment having application in the use or method of the present invention is administered as a single dose, or as an initial dose followed by a second or more subsequent doses of the antibody or its antigen-binding fragment, with the subsequent doses occurring at intervals of at least 1 day; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks. The dose may vary depending on the age and weight of the recipient, the target disease, condition, route of administration, and the like. When the antibody of the present invention is used to treat pain in adult patients, it is advantageous to administer the antibody of the present invention intravenously, subcutaneously, or intraocularly. A single dose or subsequent dose may contain an amount of the antibody or its fragment of at least 0.1 or 1 mg / kg body weight of the subject, for example, about 1 mg / kg body weight to about 100 mg / kg body weight, particularly about 10 mg / kg body weight to about 60 mg / kg body weight. Depending on the severity of endometriosis, such as the number and extent of lesions, and the degree of pain and / or comorbidities, the frequency and duration of treatment may be adjusted.

[0043] Definitions of the present invention and further embodiments, variations and alternatives: Throughout this application, the term “including” should be interpreted as encompassing all features specifically mentioned, as well as any further unspecified features of any choice. As used herein, the use of the term “including” also discloses embodiments in which there are no features other than those specifically mentioned (i.e., “consisting of” or “contributing to”).

[0044] In the context of the present invention, the terms “to treat” or “to heal” as used herein mean reducing or eliminating a lesion, preferably reducing it, or alleviating endometriosis-related pain and / or endometriosis-related comorbidities as disclosed herein.

[0045] The “therapeutic effective dose” of the antibody of the present invention is intended to be sufficient to treat the pain in a reasonable benefit / risk ratio applicable to any medical procedure. However, it should be understood that the total daily dose of the antibody should be determined by the attending physician within the bounds of sound medical judgment. The therapeutically effective dose level specific to any particular patient will depend on a variety of factors, including: the severity of endometriosis, e.g., the number and extent of lesions, and the degree of pain and / or comorbidities; the activity of the particular antibody used; the particular composition used, the woman’s age, weight, overall health, and diet; the timing, route of administration, and excretion rate of the particular antibody used; the duration of treatment; drugs used in combination with or concurrently with the particular antibody used; and similar factors well known in medicine.

[0046] "Pharmacologically" or "pharmaceutically acceptable" means molecular entities and compositions that, when administered to mammals, particularly humans, do not produce harmful, allergic, or other adverse reactions as required. A pharmacopoeia (or carrier or excipient) means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating substance, or any type of formulation aid.

[0047] The form and route of administration of the antibody-containing pharmaceutical composition of the present invention are necessarily determined by the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0048] The antibodies of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration and similar purposes. In one particular embodiment, the antibodies of the present invention are administered intravenously.

[0049] In particular, the pharmaceutical compositions comprising the antibodies of the present invention may contain a pharmaceutically acceptable medium for an injectable formulation. These may be dry, particularly lyophilized compositions, which, in particular, can be prepared by adding an isotonic and sterile saline solution (a mixture of monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium and the like or salts thereof), or optionally sterile water or saline solution, to form an injectable solution.

[0050] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow easy passage through an injection needle. It must be stable under manufacturing and storage conditions and protected from microbial contamination, such as bacteria and fungi.

[0051] The medium or carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and similar) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by coating, e.g., lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants, stabilizers, cryoprotective substances, or antioxidants. Prevention of microbial action can be achieved by antibacterial and antifungal agents. In many cases, it is preferable to include isotonic agents, e.g., sugars or sodium chloride.

[0052] Sterile injectable solutions are prepared by incorporating the required amount of the active compound, along with some of the other components listed above as needed, into a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile medium containing a basic dispersion medium and other necessary components derived from the components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any further desired components from a pre-sterilically filtered solution.

[0053] Once formulated, the solution is administered in a therapeutically effective amount in a manner appropriate to the dosage form. The formulation is readily administered in a variety of dosage forms, such as the injectable solutions described above, but drug-releasing capsules and similar forms are also available.

[0054] For parenteral administration as an aqueous solution, for example, the solution should be buffered appropriately if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, available sterile aqueous media are known to those skilled in the art in light of this disclosure. For example, a certain dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion solution, or injected into the proposed injection site (see, e.g., Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Some variation in the dose will inevitably occur depending on the condition of the person being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual.

[0055] [Table 2A]

[0056] [Table 2B]

[0057] [Table 2C]

[0058] [Table 2D]

[0059] [Table 2E]

[0060] [Table 2F]

[0061] Definitions of the present invention and further embodiments, variations, and alternatives. As used herein, a sequence "at least 85% identical to a reference sequence" is a sequence having sequence identity with respect to its total length of 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of the total length of the reference sequence.

[0062] The percentage of "sequence identity" can be determined by comparing two sequences optimally aligned across a comparison frame, where a portion of the polypeptide sequences in the comparison frame may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the same amino acid residue appears in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison frame, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison is performed by overall pairwise alignment using, for example, the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443. The percentage of sequence identity can be easily determined, for example, using the program Needle with the BLOSUM62 matrix and the following parameters: gap open = 10, gap extended = 0.5.

[0063] In the context of the present invention, "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. The conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0064] An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. Two types of light chains exist: lambda and kappa. There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains a specific sequence domain. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (collectively called CH1, CH2, and CH3). The variable regions of both the light (VL) and heavy (VH) chains determine the 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, transplacental migration, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin, consisting of a variable region with one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues derived from the hypervariable or complementarity-determining region (CDR). In some cases, residues derived from the non-hypervariable or framework region (FR) influence the overall domain structure, and therefore the binding site.

[0065] A "complementarity-determining region" or "CDR" refers to an amino acid sequence that determines both the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Therefore, the antigen-binding site of a conventional antibody contains six CDRs, including the CDR sets derived from the heavy and light chain V regions, respectively.

[0066] A "framework region" (FR) refers to an amino acid sequence inserted between CDRs, i.e., a relatively conserved portion of the immunoglobulin light and heavy chain variable region that is present among various immunoglobulins within a single species. The light and heavy chains of immunoglobulins each have four FRs, called FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively.

[0067] As used herein, “human framework region” refers to a framework region that is substantially identical (about 85%, or greater, particularly 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody.

[0068] In the context of this invention, the CDR / FR specification in the light or heavy chain of immunoglobulins will be determined based on the Kabat or IMGT specification.

[0069] Residues in antibody variable domains are traditionally numbered according to a system devised by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, United States Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used herein. Kabat residue designation does not always directly correspond to the linear numbering of amino acid residues in the sequence number sequence. Actual linear amino acid sequences, whether the framework of the basic variable domain structure or the complementarity-determining region (CDR), may contain fewer or additional amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components. The accurate Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with the "standard" Kabat-numbered sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), 50-65 (H-CDR2), and 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), 50-56 (L-CDR2), and 89-97 (L-CDR3). (http: / / www.bioinf.org.uk / abs / #cdrdef)

[0070] In the context of the present invention, the amino acid residues of the antibody of the present invention may be numbered according to the IMGT numbering system. The IMGT's unique numbering system has been established to compare variable domains regardless of the type or species of antigen receptor, chain (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, pp. 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, pp. 55-77 (2003)). In IMGT's unique numbering system, conserved amino acids always occupy the same positions, e.g., cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine, or tryptophan 118. IMGT's unique numbering system results in standardized range settings for framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: positions 39-55, FR3-IMGT: positions 66-104, and FR4-IMGT: positions 118-128) and complementarity-determining regions: CDR1-IMGT: positions 27-38, CDR2-IMGT: positions 56-65, and CDR3-IMGT: positions 105-117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the loop ends in the following order: 111, 112, 110, 113, 109, 114, etc.If the CDR3-IMGT length exceeds 13 amino acids, additional positions are created at the tip of the CDR3-IMGT loop between positions 111 and 112 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).

[0071] As used herein, the term “antibody” refers to conventional antibodies and their fragments, as well as single-domain antibodies and their fragments, in particular variable heavy chains of single-domain antibodies, and chimeric, humanized, bispecific, or polyspecific antibodies.

[0072] As used herein, antibodies or immunoglobulins also include "single-domain antibodies," which are antibodies described more recently, in which their complementarity-determining region is part of a single-domain polypeptide. Examples of single-domain antibodies include heavy-chain antibodies, naturally occurring antibodies lacking a light chain, single-domain antibodies derived from conventional quadruple-chain antibodies, and engineered single-domain antibodies. Single-domain antibodies may originate from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single-domain antibodies may be naturally occurring single-domain antibodies known as light-chain-lacking heavy-chain antibodies. In particular, species of the Camelidae family, such as camels, dromedaries, llamas, alpacas, and guanacos, naturally produce light-chain-lacking heavy-chain antibodies. Camel heavy-chain antibodies also lack the CH1 domain.

[0073] The variable heavy chains of these single-domain antibodies lacking light chains are known in the art as "VHH" or "nanobodies." Similar to conventional VH domains, VHH contains four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are approximately 10 times smaller than IgG molecules, and as a result, well-folded, functional nanobodies can be generated in vitro, while achieving high yields. Furthermore, nanobodies are highly stable and resistant to protease action. The properties and generation of nanobodies have been evaluated by Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77: pp. 13-22.

[0074] The terms “monoclonal antibody” or “mAb,” as used herein, refer to an antibody molecule with a single amino acid composition that is directed toward a specific antigen and should not be construed as requiring the production of the antibody by any particular method. Monoclonal antibodies may be produced from a single clone of a B cell or hybridoma, but may also be recombinant, i.e., produced by protein engineering.

[0075] A (conventional) antibody "fragment" includes a part of a complete antibody, particularly the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, bispecific antibodies, and bispecific and polyspecific antibodies formed from antibody fragments. Conventional antibody fragments may also be single-domain antibodies, such as heavy-chain antibodies or VHHs.

[0076] The term "Fab" refers to an antibody fragment obtained by treating IgG with the protease papain, in which approximately half of the N-terminal side of the H chain and the entire L chain are linked together via disulfide bonds, resulting in a molecular weight of approximately 50,000 Da and antigen-binding activity.

[0077] The term "F(ab')2" refers to an antibody fragment obtained by treating IgG with protease and pepsin, which is linked by disulfide bonds in the hinge region, is slightly larger than Fab, has a molecular weight of approximately 100,000 Da, and possesses antigen-binding activity.

[0078] A single-chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer typically expressed from a gene fusion containing genes encoding VH and VL, linked by a peptide-encoding linker. The human scFv fragment of the present invention contains a CDR that is maintained in an appropriate structure, particularly by using genetic recombination techniques. Bivalent and polyvalent antibody fragments may be spontaneously formed by the association of monovalent scFv, or they may be produced by linking monovalent scFv with a peptide linker such as bivalent sc(Fv)2.

[0079] "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond.

[0080] "(dsFv)2" refers to two dsFvs linked by a peptide linker.

[0081] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two antibodies within a single molecule. Therefore, a BsAb can bind to two different antigens simultaneously. Genetic engineering is increasingly being used to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described in EP2050764A1, for example.

[0082] The term "polyspecific antibody" refers to an antibody that combines the antigen-binding sites of two or more antibodies within a single molecule.

[0083] The term "bispecific antibody" refers to a small antibody fragment having two antigen-binding sites, the fragment containing a heavy-chain variable domain (VH) (VH-VL) linked to a light-chain variable domain (VL) within the same polypeptide chain. By using a linker that is too short to pair the two domains on the same chain, the domains are paired with a complementary domain on another chain, creating two antigen-binding sites.

[0084] In a particular embodiment, the epitope-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, bispecific antibodies, and VHH.

[0085] As used herein, a "chimeric antibody" is an antibody in which the constant region, or a part thereof, is altered, replaced, or exchanged, resulting in the variable region being linked to a constant region belonging to a different species or another antibody class or subclass. A "chimeric antibody" also refers to an antibody in which the variable region, or a part thereof, is altered, replaced, or exchanged, resulting in the constant region being linked to a variable region belonging to a different species or another antibody class or subclass.

[0086] The term "humanized antibody" refers to an antibody that is originally entirely or partially of non-human origin and has been modified to replace certain amino acids, particularly in the heavy and light chain framework regions, in order to avoid or minimize the immune response in humans. The constant domain of a humanized antibody is usually the human CH and CL domains. In one embodiment, the humanized antibody has a constant domain of human origin. As used herein, the term "humanized antibody" refers to a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin, such as a CDR.

[0087] The term "antibody" is used to encompass all of these types of antibodies, their fragments, or combinations.

[0088] The goal of humanization is to reduce the immunogenicity of a heterologous antibody, such as a mouse antibody, for introduction into humans while maintaining the complete antigen-binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted to avoid rejection by other mammals, may be generated using several techniques, such as resurfacing and CDR transplantation. As used herein, resurfacing techniques employ a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surface of the antibody's variable region to resemble the surface of a known antibody in the target host.

[0089] Antibodies may be humanized using a variety of other techniques, including CDR transplantation (EP0239400; WO91 / 09967; U.S. Patent Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EP0592106; EP0519596; Padlan (1991) Molecular Immunology 28(4 / 5): pp. 489-498; Studnicka et al. (1994) Protein Engineering 7(6): pp. 805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci USA 91: pp. 969-973), and chain shuffling (U.S. Patent No. 5,565,332). Human antibodies can be produced by a variety of methods known in the art, including phage display. See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Patent Applications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741.

[0090] As used herein, the term “specificity” refers to the ability of an antibody to bind to an antigen, e.g., the epitope presented in Netrin-1, to a detectable degree, but to a non-Netrin-1 protein or structure (e.g., cancer cells, or other proteins presented in other cell types) with relatively low detectable reactivity. Specificity can be determined relatively by binding or competitive binding assays, for example, using Biacore instruments, as described elsewhere herein. Specificity may be expressed by an affinity / avidity ratio of, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater, in relation to binding to a specific antigen versus nonspecific binding to other unrelated molecules (in this case, the specific antigen being Netrin-1).

[0091] The term "affinity," as used herein, refers to the strength of binding of an antibody to an epitope. Antibody affinity is given by the dissociation constant Kd, defined as [1]x[1] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [1] 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 mAb affinity are 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: pp. 589-601 (1983), which are incorporated herein by reference in their entirety. A well-known, preferred, and standard method in the art for determining the affinity of mAbs is the use of equipment from Biacore.

[0092] The present invention will now be described in more detail using the following non-limiting experiments with reference to the drawings. [Brief explanation of the drawing]

[0093] [Figure 1]This is a schematic diagram showing the timeline of the various interventions performed in this study. (A) One week (-1Wk) before surgical induction (Endo) or suturing only (Sham) of the uterine horn fragment on day 0 (d0), mice underwent ovariectomy. All mice were injected weekly with intraperitoneal (ip) estradiol benzoate (vertical red checkmarks) to maintain stable levels of circulating estrogen throughout the study. Treatment was initiated four weeks after surgical induction, and the groups consisted of Endo mice treated with NP137 (Endo + NP137 (N=15)) or control antibody NP001 (Endo + control Ab (N=16)), and Sham mice treated with control Ab NP001 (Sham + control Ab (N=16)). Bi-weekly ip treatment was maintained throughout the duration of the study. An in vivo pain assessment study comparing Sham (control Ab) and Endo (control Ab and NP137) was conducted 8–10 weeks after surgical induction (shaded box). Tissue was collected from all mice after the final behavioral assessment (diamond). The total number of mice used in this study was N=16 Sham (control Ab), N=16 Endo (control Ab), and N=15 Endo (NP137). (B) Ovariectomy was performed on mice one week (-1Wk) before inoculation with uterine horn fragments (Endo) or saline alone (Sham) on day 0 (d0). All mice were injected weekly with intraperitoneal (ip) estradiol benzoate (vertical checkmark) to maintain stable levels of circulating estrogen throughout the study. Treatment was initiated 4 weeks after surgical induction. The mice were divided into two groups: Endo mice treated with NP137 (Endo + NP137) or control antibody NP001 (Endo + control Ab (N=8)), and Sham mice treated with control Ab NP001 (Sham + control Ab (N=4)). Intravenous ip treatment was maintained every other week throughout the study. In vivo pain assessment studies comparing Sham (control Ab) and Endo (control Ab and NP137) were conducted 8–10 weeks after surgical induction (shaded boxes). Tissues were collected from all mice after behavioral assessments using the hot plate test (diamonds). The total number of mice used was N=4 Sham (control Ab), N=8 Endo (control Ab), and N=7 Endo (NP137). [Figure 2]This figure shows that NP137 treatment reduces the size of endometriotic lesions. (A1) Representative images of the reproductive system 10 weeks after induction surgery for Sham mice treated with control Ab (left box, sutures only in the uterine horn and lateral to the uterus (arrows and arrowheads indicate placement)), and Endo mice treated with either control Ab (center box) or NP137 (right box) (endometrial fragments sutured to the uterine horn and lateral to the uterus (arrows indicate placement)). (A2) Representative images of dissected and washed endometriotic lesions collected from the uterus and uterine horn of both control Ab (4 mice, left box) and Endo mice treated with NP137 (3 mice, right box). Measurement bars represent 5 mm. (B) Grouped data show a significant reduction in mean lesion mass 10 weeks after Endo induction after long-term treatment with NP137 compared to control Ab. Grouped data are shown as (B1) mean lesion mass (g) of all lesions per animal, (B2) mean lesion mass (g) of lesions located in the uterus, and (B3) mean lesion mass (g) of lesions located in the uterine horns. (C) Grouped data show a significant reduction in mean maximum lesion diameter 10 weeks after Endo induction after long-term treatment with NP137, compared to control Ab. Grouped data are shown as (C1) mean maximum lesion diameter (mm) of all lesions per animal, (C2) mean maximum lesion diameter (mm) of lesions located in the uterus, and (C3) mean maximum lesion diameter (mm) of lesions located in the uterine horns. Data represent mean ± SEM. NS, P>0.05, **P<0.01, ***P<0.001, N=9-11 mice and n=29-35 lesions per treatment group, Mann-Whitney nonparametric test for two groups with unequal variances. [Figure 3]This figure shows that NP137 treatment reduces developed vaginal hyperalgesia in endometriotic mice. (A) Grouped data showing increased visceral motor response (VMR) to vaginal distension (VD) in Endo mice treated with control Ab compared to Sham mice treated with control Ab, at 20 mmHg–70 mmHg. The increased response in Endo mice was significantly reduced at harmless distension pressures in mice treated with NP137. ^ represents Sham control Ab vs. Endo control Ab, and * represents Endo control Ab vs. Endo NP137. Data represent mean ± SEM, with * or ^P < 0.05, ** or ^^P < 0.01, **** or ^^^^P < 0.0001, generalized estimator, followed by LSD post-hoc test. The grouped data, expressed as the total area under the VMR curve for (B) all bloating pressures (20-80 mmHg), and further divided into (C) low (20-40 mmHg) bloating pressures and (D) high (60-80 mmHg) bloating pressures, show that the elevated response observed in Endo mice treated with control Ab is normalized by treatment with NP137 compared to Sham mice treated with control Ab. Data represent mean ± SEM, NS = not significant, *P < 0.05, **P < 0.01, N = 12-14 mice per treatment group, and standard one-way ANOVA with Sidak's multiple comparison post-hoc test for groups with equal variances. Each circle represents the AUC derived from the individual animal. [Figure 4]This figure shows that NP137 treatment reduces colonic hyperalgesia developed in endometriotic mice. (A) Grouped data showing increased visceral motility response (VMR) to colorectal distension (CRD) in Endo mice (yellow symbols) compared to Sham mice (blue symbols) treated with control Ab. The increase in response to CRD in Endo mice was significantly reduced in mice treated with NP137 (green symbols) between 20 mmHg and 60 mmHg. ^ represents Sham control Ab vs. Endo control Ab, and * represents Endo control Ab vs. Endo NP137. Data represent mean ± SEM, with * or ^P < 0.05, *** or ^^^P < 0.001, **** or ^^^^P < 0.0001, generalized estimator, followed by LSD post-hoc test. The grouped data, expressed as the total area under the curve of VMR for (B) all bloating pressures (20-80 mmHg), and further divided into (C) low (20-40 mmHg) bloating pressures and (D) high (50-80 mmHg) bloating pressures, show that the elevated response observed in Endo mice treated with control Ab is normalized by treatment with NP137 compared to Sham mice treated with control Ab. Data represent mean ± SEM, NS = not significant, **P<0.01, **P<0.001, ****P<0.0001, N=12-14 mice per treatment group, and Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Each circle represents the AUC derived from the individual animal. [Figure 5]This figure shows that NP137 treatment reverses the bladder dysfunction that develops in endometriotic mice. (A) Grouped data of total urine spots in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137, showing a significant increase in the total number of spots produced by Endo mice, were normalized by NP137 treatment. (B) Grouped data showing urination patterns (total random urine) divided into small (100-1,000 dpi), medium (1,001-100,000 dpi), or large (over 100,001 dpi) urine spots in Sham mice treated with control Ab (blue symbols), Endo mice treated with control Ab (yellow symbols), and Endo mice treated with NP137 (green symbols). The data shows that the significant increase in small spots produced by Endo mice is normalized by NP137 treatment. Data represent mean ± SEM, NS = not significant, *P < 0.05, **P < 0.01, N = 13-16 mice per treatment group. Standard one-way ANOVA with Tukey's multiple comparison post-hoc test was used for groups with equal variances, or Kruskal-Wallis test with Dunn's multiple comparison test was used for groups with unequal variances. Each circle represents an individual animal. [Figure 6]This figure shows that NP137 treatment reverses mechanical and thermal skin hypersensitivity developed in endometriotic mice. (A) Grouped data showing the mechanical limb retraction threshold (g) induced by electron phonfly (EvF) filament stimulation of the hindlimb in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data shows that the enhanced sensitivity to mechanical stimulation exhibited by Endo mice compared to Sham mice treated with control Ab is normalized by NP137 treatment. (B) Grouped data showing the delay (sec) in response to a heat plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data shows that the enhanced sensitivity to harmful thermal stimulation exhibited by Endo mice compared to Sham mice treated with control Ab is absent in NP137-treated Endo mice. Data represent mean ± SEM, NS = not significant, *P<0.05, **P<0.01, and ****P<0.0001, N=14-16 mice per treatment group, with standard one-way ANOVA with Tukey's multiple comparison post-hoc test for groups with equal variances, or Kruskal-Wallis test with Dunn's multiple comparison test for groups with unequal variances. Each circle represents an individual animal. [Figure 7] This figure shows that NP137 treatment improves signs of reduced overall health status in endometriotic mice during nesting. Grouped data showing nesting scores (scale 1-5) in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137 demonstrate that the reduction in nesting behavior observed in Endo mice treated with control Ab is normalized by NP137 treatment. Data represent mean ± SEM, NS = not significant, *P < 0.05, N = 14-16 mice per treatment group, and Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Each circle represents an individual animal. [Figure 8]This figure shows that NP137 treatment tends to reverse the development of thermocutaneous hypersensitivity in a syngeneic mouse model of endometriosis. The data are grouped showing the delay (seconds) in response to a heat plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data show a trend of increased sensitivity to harmful heat stimuli in Endo mice compared to Sham mice treated with control Ab, which is partially reversed in NP137-treated Endo mice. Data represent mean ± SEM, NS = not significant, N = 4-8 mice per treatment group, and standard one-way ANOVA with Tukey's multiple comparison post-hoc test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparison for groups with unequal variances. Each circle represents an individual animal. [Figure 9] This figure shows that NP137-induced improvements in the overall health status of mice with Endo are confirmed in a new cohort of animals. Grouped data showing nesting scores (scale 1-5) in sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137 demonstrate that the reduction in nesting behavior observed in Endo mice treated with control Ab is normalized by NP137 treatment. Data represent mean ± SEM, NS = not significant, *P < 0.05, N = 20-21 mice per treatment group, and Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Each circle represents an individual animal. [Figure 10]This figure shows that NP137 treatment tends to reduce the size and number of developed endometriotic lesions in a syngeneic mouse model of endometriosis. (A) Grouped data show a slight reduction in mean maximum lesion diameter 10 weeks after Endo induction after long-term treatment with NP137, compared to control Ab. (B) Grouped data show a slight reduction in mean lesion size per animal 10 weeks after Endo induction after long-term treatment with NP137, compared to control Ab. (C) Grouped data show a slight reduction in the number of developed lesions per animal 10 weeks after Endo induction after long-term treatment with NP137, compared to control Ab. Data are represented mean ± SEM. NS, P>0.05, N=5 mice and n=24-33 lesions per treatment group. Mann-Whitney nonparametric tests were used for two groups with unequal variances, and independent t-tests were used for two groups with equal variances. [Figure 11]This figure shows that NP137-induced reduction in endometriosis lesion size is confirmed in a new cohort of animals. (A1) Representative images of the reproductive system 10 weeks after induction surgery for Sham mice treated with control Ab (top panel, sutures only in the uterine horn and lateral to the uterus (arrowheads indicate placement)), and Endo mice treated with either control Ab (middle panel) or NP137 (bottom panel) (endometrial fragments sutured to the uterine horn and lateral to the uterus (arrowheads indicate placement)). (A2) Representative images of dissected and washed endometriosis lesions collected from the uterus and uterine horn of both control Ab (4 mice, top panel) and Endo mice treated with NP137 (4 mice, bottom panel). (B) Grouped data show a significant reduction in mean lesion mass 10 weeks after Endo induction after long-term treatment with NP137 compared to control Ab. Grouped data are shown as (B1) mean lesion mass (g) of all lesions per animal, (B2) mean lesion mass (g) of lesions located in the uterus, and (B3) mean lesion mass (g) of lesions located in the uterine horns. (C) Grouped data show a significant reduction in mean maximum lesion diameter 10 weeks after Endo induction after long-term treatment with NP137, compared to control Ab. Grouped data are shown as (C1) mean maximum lesion diameter (mm) of all lesions per animal, (C2) mean maximum lesion diameter (mm) of lesions located in the uterus, and (C3) mean maximum lesion diameter (mm) of lesions located in the uterine horns. Data represent mean ± SEM. NS, P>0.05, *P<0.05, **P<0.01, ****P<0.0001, N=12-16 mice and n=44-53 lesions per treatment group, with Mann-Whitney nonparametric tests for two groups with unequal variances. [Figure 12]This figure shows that the reduction in NP137-induced thermal skin hypersensitivity is confirmed in a new cohort of animals. (A) Grouped data showing the mechanical limb retraction threshold (g) induced by electron phonfly (EvF) filament stimulation of the hindlimb in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data shows that the increased sensitivity to mechanical stimulation exhibited by Endo mice compared to Sham mice treated with control Ab is normalized by NP137 treatment. (B) Grouped data showing the delay (seconds) in response to a heat plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data shows that the increased sensitivity to harmful thermal stimulation exhibited by Endo mice compared to Sham mice treated with control Ab is absent in NP137-treated Endo mice. Data represent mean ± SEM, NS = not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, N=20-22 mice per treatment group. Standard one-way ANOVA with Tukey's multiple comparison post-hoc test was used for groups with equal variances, or Kruskal-Wallis test with Dunn's multiple comparison test was used for groups with unequal variances. Each circle represents an individual animal. [Modes for carrying out the invention]

[0094] 1. Animals: The Animal Ethics Committee of the South Australian Health and Medical Research Institute (SAHMRI) approved all animal-involved experiments (Ethics No. SAM342). All animal experiments followed relevant regulatory standards and ARRIVE guidelines. Six-week-old female C57BL / 6J mice were used in SAHMRI's designated and opportunistic pathogen-free animal care facility, obtained from the in-tissue C57BL / 6J breeding program (Jax strain #000664; originally purchased from The Jackson Laboratory (Breeding Facility MP14; Bar Harbor, ME; USA)). All female mice used in this study were unmated (never mated) and housed in the absence of males from weaning. Postoperatively, mice were individually housed in individually ventilated cages (IVCs) filled with coarse chip dust-free aspen bedding (PURA®; Cat#-ASPJMAEB-CA, Niederglatt, Switzerland). The animal cages were stored on IVC shelves in the containment room in a humidity and temperature controlled environment maintaining 22±1℃ and a 12h light / 12h dark cycle. The mice had free access to LabDiet® JL rat and mouse / Auto6F diet (Cat#5K52, St. Louis, MO;USA) and were supplied with autoclaved reverse osmosis purified water.

[0095] 2. A mouse model of surgically induced endometriosis: In this study, the inventors used an adapted version of their previously established homemade mouse model of surgically induced endometriosis [9] (the entire content of this paper is incorporated herein by reference). In this study, uterine horn fragments from a total of four donors were surgically sutured lateral to the female reproductive system, with two sutures to the lateral side of the uterus (one on each side) and two sutures to the uterine horns (one on each horn). See Figure 1 for a schematic timeline of the various interventions performed in this study.

[0096] 2.1. Ovarian removal surgery: Seven days prior to surgical induction of endometriosis, female mice were ovariectomized to deplete endogenous steroid production. Briefly, before the start of surgery, the mice were anesthetized under isoflurane (2.5% / 0.5L O2) and given a low dose (0.05 mg / kg) of the analgesic buprenorphine. After sterile conditions were established, a small longitudinal incision was made in the skin and around the lumbar region of the dorsal abdominal muscles to expose the ovary on either side. With the ovary exposed, the fallopian tube / ovarian artery and vein / ovarian ligament were cut using cauterization. The uterine horn was then returned to the abdominal cavity, and the superficial skin was closed using a 9 mm clip (AutoClip® system, FST). The ovariectomized mice were allowed to recover for at least 5 days before removal of the surgical clip. Since endometriosis is an estrogen-dependent disease, it is important to maintain stable levels of circulating estrogen and minimize any differences associated with the estrous cycle stage. Taking these factors into consideration, all mice were given an intraperitoneal injection of 100 μg / kg estradiol benzoate (Progynon-B) after ovariectomy, and this was continued weekly for the duration of the study.

[0097] 2.2. Endometriosis and Sham surgery: Seven days after ovariectomy, endometriosis was surgically induced in experimental mice using uterine horn tissue collected from donor mice. To collect donor tissue, the uterine horn was excised and spread and held in place in a dissection dish containing ice-cold sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) containing penicillin (100 U / ml) and streptomycin (100 μg / ml). The uterine horn was opened longitudinally, and 4x2 mm sections of uterine horn tissue were collected using a 2 mm biopsy punch (Kai Medical, KAI00010). The recipient experimental mice were then anesthetized under isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before the start of surgery. After achieving sterility, a small midline abdominal incision was made to expose the abdominal cavity, and 2 mm circular fragments of donor uterine horn tissue were sutured to the lateral side of the uterus (2 fragments) and to the uterine horns (1 fragment per horn) using 6.0 Prolene sutures. In the Sham procedure, 6.0 Prolene sutures of similar length were sutured to the same surgical site as in the Endo procedure, but in the absence of tissue. Subsequently, the abdominal muscles were sutured closed, and the superficial skin was closed using 9 mm clips (AutoClip® system, FST). During surgery and recovery, the animals were placed on a heated mat to maintain body temperature, and complications were monitored daily. No signs of distress or abnormal pain behavior were observed during recovery.

[0098] 2.3. Mouse models of syngeneic endometriosis: In this study, the inventors used a second model of endometriosis called a syngeneic mouse model of endometriosis. This clinically relevant mouse model is produced by inoculating uterine horn fragments into the abdominal cavity, as described in a publication incorporated herein by reference

[21] . This is a minimally invasive model that resembles the spontaneous development of endometriotic lesions in humans, as the inoculated fragments are randomly dispersed throughout the abdominal cavity, where they settle and develop into lesions

[21] . This model developed both lesions and widespread chronic pelvic pain, both characteristic of endometriosis in humans.

[0099] 2.4 Endometriosis (Endo) and Sham surgery in syngeneic models: Endometriosis was induced in experimental mice by inoculating recipient mice with uterine horn tissue fragments collected from donor mice seven days after ovariectomy. To collect donor tissue, the uterine horn was excised and spread out in a dissection dish containing 0.5 ml of ice-cold sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) containing penicillin (100 U / ml) and streptomycin (100 μg / ml). The uterine horn was opened longitudinally and cut into small fragments. Next, before initiating induction, recipient experimental mice were anesthetized under isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine. After sterilization, a small midline abdominal incision was made to expose the abdominal cavity, and half of the collected donor uterine horn tissue fragments were inoculated into the abdominal cavity of one recipient mouse using a 1 ml pipette (a total volume of solution containing the fragments was approximately 0.2 ml). In the Sham procedure, a similar procedure was followed, except that 0.2 ml of sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) was inoculated instead. Following this, the abdominal muscles were sutured closed, and the skin surface was closed using 9 mm clips (AutoClip® system, FST). During surgery and recovery, the animals were placed on a heated mat to maintain body temperature, and daily monitoring for complications was performed. No signs of distress or abnormal pain behavior were observed during recovery.

[0100] 3. Characterization of endometriotic lesion proliferation and development: Endometriotic lesions proliferating from surgically transplanted uterine horn fragments were collected and measured at the completion of all behavioral assays to track endometriotic lesion development after treatment with either control Ab or NP137. To minimize any acute effects of either treatment, all tissue was collected at least 24 hours after the last control Ab or NP137 treatment, and at least 3 days after estradiol administration to minimize estrogen changes between time points.

[0101] 3.1 Collection and measurement of endometriotic lesions: Lesions from Endo (control Ab) and Endo (NP137) mice were locibly located, dissected, measured, and weighed after death. A total of four endometriotic lesions were collected from each mouse (2x uterine and 2x uterine horn lesions). Lesions damaged during dissection or not cleanly isolated from surrounding tissue were not included in the lesion growth analysis. Measurements were taken at the widest point of the lesion using a caliper (Castroviejo caliper 8.5 cm / 3-1 / 4").

[0102] 3.2. Statistical analysis of lesion growth: Data are presented as mean ± SEM, where N represents the number of animals and n represents the number of individual lesions per group. Data were graphed using Prism9 software (GraphPad Software, San Diego, CA, USA). For two groups with unequal variances, the data were statistically analyzed using the Mann-Whitney nonparametric test. ** P<0.01, *** P<0.001, **** We considered the difference to be statistically significant when P < 0.0001.

[0103] 4. In vivo evaluation of chronic pelvic pain by quantifying visceral motor response (VMR). Visceral motility response (VMR) is a nociceptive brainstem reflex consisting of abdominal muscle contractions in response to adverse distension of lumen organs, such as the vagina and colorectal tract [4, 6, 7, 19, 25, 27, 16]. The inventors recorded VMR for vaginal distension (VD) or colorectal distension (CRD) as an objective measure of vaginal and colonic sensitivity to pain in fully conscious animals [14, 3, 22, 24, 5, 2, 8]. Using this technique, the inventors previously showed that mice with endometriosis develop both colonic and vaginal hypersensitivity [9, 21]. To determine whether long-term treatment with NP137 can reduce vaginal and colonic hypersensitivity developed in a mouse model of endometriosis, the inventors used VMR to determine the difference between Sham (control Ab) and Endo mice (control Ab or NP137). To minimize any acute effects of any treatment, all mice were studied at least 24 hours after the last control Ab or NP137 treatment, and at least 3 days after estradiol administration to minimize estrogen changes between time points.

[0104] 4.1. Surgical implantation of electromyography (EMG) electrodes: VMR is objectively evaluated by recording the electrical activity (electromyography (EMG)) generated by abdominal muscle contractions in response to harmless and harmful vaginal or colorectal distension. To enable this, three days prior to VMR, under isoflurane anesthesia, the exposed ends of two Teflon-coated stainless steel wires (Advent Research Materials Ltd, Oxford, UK) were sutured to the right external oblique muscle of Sham or Endo mice, and subcutaneously pierced so as to be exposed at the base of the neck for future use. As previously described [21, 9, 8], at the end of the surgery, the mice were administered prophylactic antibiotics (Baytril®; 5 mg / kg sc) and analgesics (buprenorphine; 0.5 mg / 10 kg sc), returned to their individual habitats, and allowed to recover for at least three days.

[0105] 4.2. Evaluate the visceral motility response (VMR) to vaginal distension (VD) or colorectal distension (CRD): As previously described [21, 9, 6, 7], on the day of VMR evaluation, mice were temporarily anesthetized with inhaled isoflurane, administered physiological saline enema via catheter into either the colorectum (100 μL) or vaginal cavity (50 μL), and a balloon was inserted into either the colorectum or vaginal cavity for VMR evaluation. Briefly, the lubricated balloon was gently introduced through the anus for the CRD and inserted into the colorectum to 0.25 cm past the anal margin (2.5 cm long latex balloon), and gently passed through the vagina for the VD and inserted to 1 mm proximal to the vaginal margin (3 mm long latex balloon). The balloon catheter was secured to the base of the tail with surgical tape and connected to a barostat (Isobar 3, G&J Electronics, Willowdale, Canada) for gradual and pressure-controlled balloon inflation. Mice were gently restrained in a mouse restraint that allowed access to the dorsal side, and recovered from anesthesia for 10 minutes prior to the start of the distension flow. The distension flow was pre-set and applied by barostat to 20-40-50-60-70-80 mmHg (duration 20 seconds) at 4-minute intervals for the CRD, and to 20-30-40-60-70-80 mmHg (duration 30 seconds) at 3-minute intervals for the VD. As previously described [6], for offline analysis using Spike2 (Cambridge Electronic Design), EMG electrodes were relayed to the data acquisition system, where the signal was recorded (NL100AK headstage), amplified (NL104), filtered (NL 125 / 126, Neurolog, Digitimer Ltd, bandpass 50-5000Hz), and digitized for a PC (CED1401, Cambridge Electronic Design, Cambridge, UK). Mice were allowed to recover from the initial distension protocol (randomized to either VD or CRD) for at least 3 hours before the next distension protocol. After this final distension, the mice were returned to IVC cages and then returned to the shelves for final ip treatment and tissue collection.

[0106] 4.3. Statistical analysis of VMR against VD and CRD Analog EMG signals were rectified, integrated, and used for VMR analysis. To quantify the magnitude of VMR at each inflation pressure, the area under the curve (AUC) during inflation was corrected for baseline activity (AUC before inflation). AUC was quantified by calculating the AUC of the EMG signal at each inflation pressure. Furthermore, the sum of AUCs for all combined inflation pressures (total AUC) was also analyzed as an overall parameter of the VMR response for each individual mouse. VMR data are presented as mean ± SEM, where N represents the number of animals. Analysis and numerical data were prepared using GraphPad Prism software (version 9, San Diego, CA, USA). VMR data for AUC at each inflation pressure were statistically analyzed using generalized estimating equations (GEE) followed by minimum significance (LSD) post-hoc tests, using SPSS 23.0 where appropriate. Data for total AUC were statistically analyzed using Prism9 software (GraphPad Software, San Diego, CA, USA). Where appropriate, groups with equal variances were analyzed using a standard one-way ANOVA with Sidac's multiple comparison post-hoc test, or groups with unequal variances were analyzed using Dunn's Kruskal-Wallis test with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001, **** We considered the difference to be statistically significant when P < 0.0001.

[0107] 5. In vivo evaluation of skin sensitivity induced by thermal and mechanical stimuli. To determine whether long-term treatment with NP137 could reduce the elevated induced pain-like behaviors developed in a mouse model of endometriosis, the inventors assessed skin sensitivity using both a hot plate (heat sensitivity) and an electron phonfly (EvF) test (mechanical sensitivity). To ensure consistency and reduce bias, all measurements were blinded and performed by the same researchers. All mice were studied at least 24 hours after the last control Ab or NP137 treatment to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen changes between time points.

[0108] 5.1. Evaluate skin sensitivity to mechanical stimuli using the electron phonfly (EvF) reflex: The electronic phonfly (EvF) test is used to assess mechanical allodynia to mechanical stimuli by measuring the retraction of the limb in response to stimuli that are not normally painful

[12] . The inventors have previously used this technique to demonstrate the development of mechanical sensitivity in a mouse model of endometriosis [9]. Prior to the EvF test, mice were acclimated to a transparent plexiglass enclosure (observation chamber approximately 230 x 240 x 146 mm, BSBIOEVF, Panlab, Spain) and placed on a raised wire mesh platform for 30 minutes daily for more than two days. On the day of the test, the mice were moved to the test chamber in the IVC and acclimated for at least 15 minutes. The animals were placed individually within the test area, with the vent cover in place, and left for 15 minutes. A semi-flexible tip was attached to the EvF unit (handheld force transducer, BSBIOEVF4s, Panlab, Spain) and set to zero. When the animals were still and quiet, the force transducer was applied perpendicularly to the animal's hind limb or lower abdomen from below. The force was gradually increased until a clear retraction response was observed. The maximum applied force (grams) that induced retraction of the limb or abdomen was recorded as the retraction threshold. Each region was stimulated five times, and the average was recorded as the experimental retraction threshold for each mouse. At the end of the experiment, the mice were returned to their living cages and then to the IVC shelves.

[0109] Statistical analysis: The average force in grams required to elicit a withdrawal response using EvF was recorded for analysis. Data are presented as mean ± SEM, and N represents the number of animals. Data were statistically analyzed using Prism 9 software (GraphPad Software, San Diego, CA, USA), and where appropriate, ordinary one-way ANOVA with Tukey's multiple comparison post-test for groups with equal variance; or Kruskal-Wallis test with Dunn's multiple comparison for groups with unequal variance was used for analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 was considered statistically significant for the difference.

[0110] 5.2. Evaluating skin sensitivity to noxious heat stimuli by hot plate latency: The hot plate test is a common test used to measure acute thermal nociception by monitoring reflexive behavior to a heat stimulus. Using this technique, the inventors have previously shown the development of thermal hypersensitivity in a mouse model of endometriosis [21, 9]. Prior to the test, mice were transferred to the laboratory in an IVC and acclimated for at least 15 minutes. Then the mice were individually placed on a pre-heated hot plate (LE7406, Harvard Apparatus, Panlab, Spain) set at 54 ± 1 °C, which has been shown to be a noxious stimulus

[12] , and surrounded by a transparent methacrylate protective casing. The time between being placed on the hot plate and the first sign of a pain response, including licking the hindlimb, quickly moving the hindlimb, or jumping up (whichever occurred first), was recorded as the hot plate latency. Mice were allowed to recover in the cage for 24 hours and the test was repeated. The average response latency for both tests was recorded as the final hot plate latency. Then the mice were returned to their home cages and IVC shelves.

[0111] Statistical analysis: The mean response in seconds obtained from both exposures to the heat plate was recorded for analysis. Data are presented as mean ± SEM, and N represents the number of animals. Data were statistically analyzed using Prism9 software (GraphPad Software, San Diego, CA, USA), and the Kruskal-Wallis test with Dunn's multiple comparisons was used for groups with unequal variances. * P<0.05, ** P<0.01, *** P<0.001, **** We considered the difference to be statistically significant when P < 0.0001.

[0112] 6. In vivo evaluation of spontaneous behavior: To determine whether long-term treatment with NP137 could reduce the exacerbation of spontaneous pain-like behaviors that occurred in a mouse model of endometriosis, the inventors used urination pattern analysis along with overnight nesting analysis to determine the difference between Sham (control Ab) and Endo mice (control Ab or NP137). To minimize any acute effects of either treatment, all mice were evaluated at least 24 hours after the last control Ab or NP137 treatment, and at least 3 days after estradiol administration to minimize estrogen changes between time points. To ensure consistency and reduce bias, all measurements were blinded and performed by the same researchers. At the end of each test protocol, mice were returned to their living cages and then to the IVC shelves for subsequent assays.

[0113] 6.1 In vivo assessment of bladder dysfunction by voiding pattern analysis: Voiding pattern analysis is a voiding assessment tool that provides information about changes in spontaneous behavioral patterns associated with urinary tract pathologies, including overactive bladder (OAB) and interstitial cystitis / painful bladder syndrome (IC / PBS) [16, 17]. Voiding pattern analysis was performed as previously described to evaluate changes in bladder function after the development of endometriosis in mice [9, 21].

[0114] Eight weeks after endometriosis or sham-inducing surgery, and four weeks after either control Ab or NP137 treatment, urination patterns were analyzed in all mice. For this purpose, all bedding was removed from the cages of each individual Endo and Sham mouse, and filter paper was placed at the bottom of each cage. Between 9 AM and 12 PM, the mice were placed in the arranged cages for three hours with free access to food and water. During this time, the mice produced spontaneous urination patterns on the filter paper. The filter paper was collected and stored for imaging, the bedding was returned to the IVC, and the mice were returned to the IVC shelves.

[0115] Urine collected using filter paper was imaged using a UV transilluminator (Bio-Rad, California, USA) and digitized into binary images using ImageJ software (NIH, ImageJ, 2.0.0). The number and size of urine spots were determined using pre-set thresholds in the ImageJ software, and for this study, the number of small (100–1,000 pixels), medium (1,001–100,000 pixels), and large (over 100,000 pixels) spots was quantified.

[0116] Statistical analysis: Data are presented as mean ± SEM, where N represents the number of animals. Data were graphed using Prism9 software (GraphPad Software, San Diego, CA, USA), and analysis was performed using, where appropriate, a standard one-way ANOVA with Tukey's multiple comparison post-hoc test for groups with equal variances, or the Kruskal-Wallis test with Dunn's multiple comparison test for groups with unequal variances. * P<0.05, ** P<0.01, *** P<0.001, **** We considered the difference to be statistically significant when P < 0.0001.

[0117] 6.2. Assessment of overall animal health status through analysis of spontaneous overnight nesting: Spontaneous nesting analysis is a sensitivity assessment tool that provides information about changes in spontaneous behavioral patterns related to overall health in mice

[13] . In this study, as previously described

[11] , we performed an analysis of overnight nesting using a predefined 5-point scale to evaluate the long-term effects of NP137 administration on overall animal health.

[0118] Eight weeks after endometriosis or sham induction surgery, and four weeks after either control Ab or NP137 treatment, nesting pattern analysis was performed on all mice. For this purpose, all environmental reinforcement items (including the original nesting material, cardboard tunnels, and tissue) were removed from the living cages of each individual Endo and Sham mouse, leaving only the original bedding material at the bottom of each cage. Pre-weighed nesting materials (approximately 3g of 5cm square cotton wafers) were placed in each cage, and the mice were returned to the IVC shelf and left to nest overnight (4 PM to 9 AM). As previously described

[11] , the following morning, each nest was evaluated blindly and graded according to a final predetermined 5-point nesting rating scale ranging from 1 to 5, where a score of 1 reflected no contact with the nesting material, and a score of 5 represented a completely torn nesting material with high, fluffy walls. The environmental reinforcements were returned to the living cages, and the mice were returned to the IVC shelf.

[0119] Statistical analysis: Data are presented as mean ± SEM, where N represents the number of animals. Data were graphed using Prism9 software (GraphPad Software, San Diego, CA, USA) and analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001, **** We considered the difference to be statistically significant when P < 0.0001.

[0120] 7. Results and Conclusions: 7.1. Mice with endometriosis developed cystic endometriotic lesions: The mouse model of endometriosis used in this study is a modification of a previously established model of surgically transplanted endometriosis [9]. In this model, the inventors transplanted uterine horn fragments to each side of the upper part of the uterus and to the ends of the uterine horns, as detailed in the Methods section (Figure 2A1 as well). Control mice (Sham mice) were generated using the same surgical procedure, but in the absence of uterine horn tissue (Figure 2A1). As previously described [9], the transplanted fragments developed into endometriotic lesions 10 weeks after transplantation (Figure 2A1 as well).

[0121] 7.2. NP137 treatment reduced the development of endometriotic lesions. To investigate whether NP137 treatment can limit lesion development and provide analgesia in mice with endometriosis, the inventors examined the proliferation of endometrial lesions. The effectiveness of NP137 treatment was quantified by comparing the degree of endometrial development in Endo mice treated with NP137 to the degree of endometrial development observed in Endo mice treated with the control antibody NP001 (control Ab) (Figure 2A1). As previously described, Sham mice do not develop endometrial lesions at the surgical suture site (Figure 2A1).

[0122] Examination of the female reproductive systems of mice with endometriosis showed that long-term NP137 treatment reduced the size of developed lesions (Figures 2A1-2A2). The reduction in lesion size is visually evident in Figure 2A2, which shows endometrial lesions collected from four Endo mice treated with NP001 (control Ab) (Figure 2A2, left panel) and endometrial lesions collected from three Endo mice treated with NP137 (Figure 2A2, right panel). Quantitative analysis of the collected lesions showed that NP137 treatment significantly reduced both the mass and size of these endometriotic lesions (Figures 2B-2C). Interestingly, the NP137-induced reduction in lesion size was specific to lesions that developed around the uterus but not to lesions that proliferated at the terminal uterine horns (Figures 2A2 and 2B-2C). Surprisingly, a separate series of treatments (four more Endo mice treated with NP001 and five more Endo mice treated with NP137) provided the first evidence of NP137's ability to significantly reduce lesion size in mice with Endo (Figures 11A1-11A2). Furthermore, by including the data from these additional mice into the overall dataset (Figures 11B-11C), the regional differences observed in the initial series of treatments were eliminated (Figures 2A2 and 2B-2C). Therefore, NP137 is efficient in reducing endometriotic lesion development regardless of the location where the lesions developed (Figures 11B2-11B3 and 11C2-11C3).

[0123] Interestingly, the inventors also tested the effect of NP137 on reducing lesions developed in different mouse models of endometriosis, known as syngeneic mouse models of endometriosis (Figure 10). This is a minimally invasive model that resembles the spontaneous development of endometriotic lesions in humans, as the inoculated fragments are randomly dispersed throughout the peritoneal cavity, where they settle and develop into lesions. Surprisingly, NP137 treatment tended to reduce the size and number of lesions developed in the peritoneal cavity of syngeneic mouse models of endometriosis (Figures 10A-10C). This suggests that NP137 is not only efficient at reducing the proliferation of lesions once they have formed, but may also inhibit the formation of lesions themselves.

[0124] 7.3. NP137 treatment reduced developed vaginal hypersensitivity in endometriosis. Visceral pain was measured in vivo by recording the visceral motor pain response (VMR) induced by vaginal distension (VD). VMR is an absolute standard for measuring pain originating from tubular organs, such as the vagina [3, 26] and the colon (as in our previous studies [6, 16]). As previously described [8], the VMR response was quantified by recording electromyographic (EMG) activity derived from electrodes surgically implanted in the abdominal muscles of mice.

[0125] In Sham mice, vaginal distension increased the VMR response, and the degree of VMR was related to the applied pressure (Figure 3A). We found that mice with fully developed endometriosis showed a significant increase in the VMR response to VD of 20 and 40-70 mmHg (Figure 3A), and all combined distensions (Figure 3B), indicating the development of vaginal hyperalgesia in endometriotic mice. Surprisingly, compared to Endo mice treated with control Ab, treatment with NP137 significantly reduced the VMR response in Endo mice, especially at lower distension pressures (less than 40 mmHg) (Figure 3C). Furthermore, the overall effect of NP137 normalized the VMR response to the level shown in Sham control mice (Figure 3D).

[0126] Overall, these results suggest that treatment with the monoclonal antibody NP137 is an effective approach to alleviate vaginal hyperdysorpticity associated with endometriosis.

[0127] 7.4. NP137 treatment reduced developed colorectal hyperalgesia in endometriosis. Women with endometriosis are usually diagnosed with other long-term visceral comorbidities, such as irritable bowel syndrome (IBS)

[29] . The inventors then aimed to determine whether mice with fully developed endometriosis also have increased pain sensitivity induced by colorectal distension (CRD) and whether NP137 treatment can provide analgesia.

[0128] The inventors found that Endo mice treated with control Ab showed significant hypersensitivity to all CRDs of tested distensions between 20 and 80 mmHg (Figure 4A), and all combined distensions (Figure 4B), compared to Sham mice treated with control Ab, indicating the development of colorectal hyperalgesia in endometriotic mice. Similar to what was observed in VD, treatment with NP137 significantly reduced the VMR response in Endo mice compared to Endo mice treated with control Ab, particularly at lower distension pressures (less than 40 mmHg) (Figure 4C). Furthermore, the overall effect of NP137 normalized the VMR response to the level shown in Sham control mice (Figure 4D).

[0129] Importantly, these findings are clinically relevant because women with endometriosis often experience severe colorectal hyperalgesia involving both the colon and vagina [20, 29]. Overall, these results suggest that treatment with the monoclonal anti-netrin antibody, NP137, is an effective approach to alleviating colorectal hyperalgesia associated with endometriosis.

[0130] 7.5. NP137 treatment normalized the bladder function changes that had developed in endometriosis. Women with endometriosis often suffer from visceral comorbidities, including overactive bladder (OAB) and interstitial cystitis / painful bladder syndrome (IC-PBS). In this study, the inventors aimed to determine whether their mouse model of endometriosis develops altered bladder function, and, importantly, whether NP137 treatment can normalize that function. To investigate this, the inventors examined and compared the spontaneous bladder-voiding patterns of Sham and Endo mice treated with control Ab to those of Endo mice treated with NP137, as an indicator of altered bladder function.

[0131] The inventors found that mice with fully developed endometriosis exhibit altered bladder voiding patterns compared to sham mice, as evidenced by the increased total number of urinary spots (Figure 5A). This disordered, scattered voiding pattern, characterized by an increased number of small urinary spots (Figure 5B), indicates urgent and frequent OAB / IC-PBS symptoms

[17] .

[0132] Interestingly, treatment with NP137 normalized the bladder dysfunction developed in mice with endometriosis, as evidenced by the reduction in the total number of urinary spots (Figure 5A), particularly small urinary spots (Figure 5B), observed in NP137-treated Endo mice. Furthermore, the overall effect of NP137 normalized the urination pattern to the same extent as that shown in Sham control mice (Figures 5A-5B).

[0133] 7.6. NP137 treatment normalizes the increased skin sensitivity to heat and mechanical stimuli that develops in endometriosis. In addition to comorbidities related to internal organs, women with endometriosis may also experience changes in skin sensitivity [18, 28]. Here, we determine whether our mouse model of endometriosis exhibits changes in skin sensitivity and whether NP137 treatment can cure it. To determine whether the development of endometriosis affects skin sensitivity induced by heat and mechanical stimuli, mice were subjected to hot plate and electron fonfry (EvF) tests.

[0134] The inventors found that mice with fully developed endometriosis treated with control Ab exhibited enhanced sensitivity to mechanical stimulation of the hind limbs, indicated by a significant reduction in the force required to elicit a response compared to Sham mice (Figure 6A). The force required to elicit a response is called the mechanical threshold, and a reduction in this threshold indicates enhanced sensitivity to mechanical stimulation. The inventors also found that Endo control Ab-treated mice exhibited enhanced sensitivity to thermal stimulation, indicated by a significant reduction in the delay of response to a hot plate compared to Sham mice (Figure 6B). These results demonstrate that the inventors' mouse model of endometriosis actually experiences enhanced sensitivity to mechanical and thermal stimuli to the skin compared to Sham mice.

[0135] Interestingly, the inventors found that treatment with NP137 efficiently reversed the increased sensitivity to both mechanical and thermal stimuli observed in Endo mice treated with control Ab (Figures 6A-6B). Importantly, the reduction observed was at a similar level to that observed in Sham mice treated with control Ab (Figures 6A-6B). Surprisingly, a separate series of treatments (six more Endo mice treated with NP001 and six more Endo mice treated with NP137) confirmed the first findings demonstrating NP137's ability to significantly reduce both mechanical and thermal hypersensitivity of the skin in mice with Endo (Figures 12A-12B).

[0136] Furthermore, the inventors found that mice with Endo induced by inoculation of uterine horn fragments into the abdominal cavity (syngeneic mouse model) also exhibited individual enhancements in sensitivity to thermal stimulation, indicated by individual reductions in the delayed response to a hot plate compared to the Sham equivalent (Figure 8). Interestingly, the inventors found that treatment with NP137 tended to reverse this effect observed in Endo mice treated with control Ab (Figure 8).

[0137] 7.7.NP137 improves signs of overall health decline shown in endometriosis. Finally, the inventors tested whether the development of endometriosis alters spontaneous behavior in a manner that indicates a deterioration in the animals' overall health, and whether NP137 treatment can help maintain the animals' overall health. Spontaneous nesting analysis is a sensitivity assessment tool that provides information about changes in spontaneous behavior patterns related to overall health in mice

[13] .

[0138] In this study, the inventors analyzed the overnight nesting behavior of Sham and Endo mice treated with control Ab and found that mice with fully developed endometriosis exhibited reduced nesting ability during this period (Figure 7). This reduction in nesting indicates a lack of nocturnal activity, suggesting that the animals' health status is diminished by the development of endometriosis. The inventors found that treatment with NP137 partially restored nesting behavior in mice with endometriosis (Figure 7), and that Endo mice treated with NP137 exhibited nesting behavior similar to that of Sham mice (Figure 7). Surprisingly, a separate series of treatments (six more Endo mice treated with NP001 and five Endo mice treated with NP137) confirmed the first findings demonstrating NP137's ability to significantly improve the overall health status of mice with Endo (Figure 9). By incorporating data from these additional mice into the overall dataset, it was shown that NP137 treatment completely reversed the absence of overnight nesting that occurred in mice with Endo. This was evident from the fact that mice with Endo treated with NP137 exhibited nesting behavior patterns similar to those of Sham mice treated with NP001 (Figure 9), demonstrating the beneficial effect of NP137 on reducing anxiety-like behaviors associated with endometriosis.

[0139] 7.8. Conclusion Overall, the results of this preclinical trial demonstrate that long-term treatment with NP137, a clinically approved mAb anti-netrin-1, is effective in reducing the size of developed endometriotic lesions in clinically relevant mouse models of endometriosis. Furthermore, NP137 treatment reversed altered pain sensitivity across multiple peripheral organs and reduced chronic pelvic pain experienced by mice with fully developed endometriosis. These results strongly suggest that NP137 is a promising therapeutic strategy for reducing or eliminating endometriotic lesions and alleviating chronic pelvic pain in women with endometriosis. 8. (References): TIFF2026510571000008.tif212157TIFF2026510571000009.tif233155TIFF2026510571000010.tif150155

Claims

1. An anti-Netrin-1 antibody or its antigen-binding fragment for use in treating endometriosis, wherein the treatment includes reduction of endometriotic lesions, particularly uterine and / or uterine horn endometriotic lesions.

2. The antibody or antigen-binding fragment thereof for use according to claim 1, further comprising the treatment of relieving chronic pelvic pain.

3. The antibody or antigen-binding fragment thereof for use according to claim 1 or 2, wherein the treatment further comprises alleviating vaginal hyperalgesia.

4. The aforementioned treatment is: - Reduction of colorectal hyperalgesia developed in endometriosis; - Improvement of bladder dysfunction that has developed in endometriosis; - Reduction of increased skin sensitivity to heat and / or mechanical stimuli that develops in endometriosis; - Improvement of overall health status altered by endometriosis An antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 3, further comprising:

5. An anti-Netrin-1 antibody or antigen-binding fragment for use according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment specifically binds to a polypeptide of the sequence of SEQ ID NO:

33.

6. The antibody is a monoclonal antibody or its antigen-binding fragment, and the antibody or fragment is - H-CDR1 having the sequence described in Sequence ID No. 5, - H-CDR2 having the sequence described in Sequence ID No. 6, - H-CDR3 having the sequence described in Sequence ID No. 7 Variable domain VH, - L-CDR1 having the sequence described in Sequence ID No. 8, - L-CDR2 having sequence YAS, - L-CDR3 having the sequence described in Sequence ID No. 9 Variable domains (VL) including or - H-CDR1 having the sequence described in Sequence ID No. 28, - H-CDR2 having the sequence described in Sequence ID No. 29, - H-CDR3 having the sequence described in Sequence ID No. 30 Variable domain VH, - L-CDR1 having the sequence described in Sequence ID No. 31, - L-CDR2 having the sequence described in Sequence ID No. 32, - L-CDR3 having the sequence described in Sequence ID No. 9 Variable domain (VL) An anti-Netrin-1 antibody or its antigen-binding fragment for use according to any one of claims 1 to 5, comprising:

7. An anti-Netrin-1 antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 6, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, and the antibody or fragment thereof comprises a pair of VH and VL sequences selected from the following pairs: SEQ ID NOs: 27 and 19, SEQ ID NOs: 20 and 14, SEQ ID NOs: 21 and 15, SEQ ID NOs: 22 and 16, SEQ ID NOs: 23 and 17, SEQ ID NOs: 24 and 17, SEQ ID NOs: 25 and 16, SEQ ID NOs: 26 and 17, preferably the antibody or fragment thereof comprises SEQ ID NOs: 22 and 16, which are a pair of VH and VL sequences.

8. An anti-netrin-1 antibody for use according to any one of claims 1 to 7, comprising a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), particularly a human kappa constant domain.

9. An antibody for use according to any one of claims 1 to 8, comprising VH of the sequence of SEQ ID NO: 22 and VL of the sequence of SEQ ID NO:

16.

10. The antibody for use according to claim 9, further comprising human IgG1 CH Genbank AEL33691.1 modified R97K and human IgG1 CL kappa Genbank CAC20459.1.

Images