USE OF AN ANTI-NETRIN-1 ANTIBODY OR AN ANTIGEN-BINDING FRAGMENT THEREOF

Abstract

The invention concerns the treatment of endometriosis using an anti-netrin- 1 antibody or an antigen-binding fragment thereof, and such antibody or fragment thereof for use in treating endometriosis. Said treating comprises a reduction of the endometriosis lesions, especially the uterine and / or uterine horn endometriosis lesions. Said treating further comprises a chronic pelvic pain relief or vaginal hyperalgesia relief. It may also comprise a reduction of colorectal hyperalgesia developed in endometriosis, an improvement of bladder dysfunction developed in endometriosis, a reduction of enhanced cutaneous sensitivity to thermal and / or mechanical stimuli developed in endometriosis, an improvement of the overall wellbeing altered by the endometriosis.

Description

1 / 64 “USE OF AN ANTI-NETRIN-1 ANTIBODY OR AN ANTIGEN-BINDING FRAGMENT THEREOF”

[001] The present invention relates to medicines and methods for the treatment of endometriosis, allowing a reduction or disappearance of endometriosis lesions and, more specifically, combining a favorable therapeutic effect on endometriosis and the lesions that mark it, and on the associated pain(s), including vaginal hyperalgesia and on other comorbidities. Background of the Invention

[002] Endometriosis is a chronic inflammatory gynecological disease affecting approximately 10% of women of reproductive age, generating an economic burden of nearly US$80 billion / year in the US alone. Chronic pelvic pain (CPP) is the most debilitating symptom experienced by women with endometriosis. Furthermore, patients with endometriosis are commonly co-diagnosed with visceral comorbidities, including irritable bowel syndrome (IBS) and overactive bladder syndrome (OBS). Current research on the underlying mechanisms of pain in endometriosis focuses primarily on endometriotic lesions and their inflammatory environment as a source of CPP. However, surgical removal of endometriotic lesions does not provide reliable pain relief, and adequate treatments are still lacking.

[003] According to the World Health Organization, endometriosis is a disease characterized by the presence of tissue similar to the endometrium (the lining of the uterus) outside the uterus. It causes a chronic inflammatory reaction that can result in the formation of scar tissue (adhesions, fibrosis) in the pelvis and other parts of the body. Several types of lesions have been described, such as superficial endometriosis, found mainly in the pelvic peritoneum, cystic ovarian endometriosis (endometrioma), found in the ovaries, deep endometriosis, found in the rectovaginal septum, bladder and bowel, and Petition 870250075665, dated 08 / 26 / 2025, pages 165 / 234 2 / 64 In rare cases, endometriosis has also been found outside the pelvis. Symptoms associated with endometriosis vary and include a combination of painful menstrual periods, chronic pelvic pain, pain during and / or after sexual intercourse, painful bowel movements, pain when urinating, fatigue, depression or anxiety, abdominal distension, and nausea.

[004] Currently, there is no known cure for endometriosis. Treatment generally aims to control symptoms, which is clearly insufficient.

[005] There is therefore a need for new therapeutic treatments capable of reducing, limiting or reversing lesions resulting from endometriosis and, preferably, at the same time, capable of reducing hyperalgesia or pain related to endometriosis and possibly also other comorbidities or symptoms of comorbidity. Brief Description of the Invention

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

[23] , has demonstrated anticancer effects in preclinical mouse models

[15] and is currently in Phase II clinical trials for advanced-stage cancer (https: / / clinicaltrials.gov / ct2 / show / NCT02977195). It is shown here for the first time that NP137 has in vivo the unusual and surprising ability to reduce both the development of endometriosis lesions and chronic pelvic pain associated with endometriosis, providing relief from vaginal hyperalgesia. Compelling data were obtained in two well-characterized mouse models of endometriosis exhibiting symptoms similar to those observed in women with endometriosis (i.e., the development of endometrial lesions and generalized chronic pelvic pain).

[007] In one aspect, the invention relates to an antinetrin-1 antibody or an antigen-binding fragment thereof, or a Petition 870250075665, dated 08 / 26 / 2025, pp. 166 / 234 3 / 64 pharmaceutical composition containing an anti-netrin-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier, for use in the treatment of endometriosis, said treatment comprising a reduction of endometriosis lesions, especially uterine and / or uterine horn endometriosis lesions.

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

[009] In another embodiment, the anti-netrin-1 antibody and its antigen-binding fragment are defined below with the disclosed set of 6 CDRs, or the disclosed specific VH and VL regions.

[0010] In preferred embodiments, the anti-netrin1 antibody and the antigen-binding fragment comprise the HV and LV pairs of any of the humanized monoclonal antibodies HUM01-10.

[0011] In a preferred embodiment, the antinetrin-1 antibody is NP137.

[0012] In an additional aspect, the aforementioned treatment comprises a reduction in both endometriosis lesions developed within the peritoneal cavity (i.e., around the uterus and / or uterine horns) and relief of chronic pelvic pain in endometriosis.

[0013] In particular, the aforementioned treatment comprises the reduction of endometriosis lesions and / or hemorrhagic lesions. Through imaging, it is possible to observe, measure, and take into account the reduction in the total volume of the lesions and / or the reduction in the volume of the hemorrhagic lesions. We consider a reduction to be significant when the total volume of the lesions is reduced by more than 20%. In one embodiment, the antibody of Petition 870250075665, dated 08 / 26 / 2025, pp. 167 / 234 4 / 64 invention allows a reduction in lesion size of more than 20, 30, 40 or 50%. Imaging can be performed using imaging methods used for endometriosis, such as transvaginal ultrasound, magnetic resonance imaging (MRI; for more details, see

[30] ) and computed tomography.

[0014] Furthermore, treatment with NP137 results in improvement of vaginal hyperalgesia developed in endometriosis, as assessed in our model by means of electromyographic (EMG) signal analysis of visceromotor responses (VMR) evoked by vaginal distension (VD), as described in section 4. In women, we used the VAS (visual analog scale). A treatment is generally considered to have a certain degree of efficacy in painful endometriosis when pain scales show a reduction of at least 2 points on the VAS (out of 10). In one embodiment, the antibody of the invention allows a reduction of at least 2, 3 or 4 points on the VAS. For more details on the VAS, see

[10] .

[0015] In other respects, the aforementioned treatment may also include one or more of the following additional technical effects or therapeutic functions: - a reduction in colorectal hyperalgesia developed in endometriosis, - a 75% improvement in the recovery from bladder dysfunction developed in endometriosis, - a reduction in the increased skin sensitivity to thermal stimuli developed in endometriosis, - a reduction in the increased skin sensitivity to mechanical stimuli developed in endometriosis, - Improvement in overall well-being that had been altered by endometriosis.

[0016] In one aspect, the invention relates to a method of Petition 870250075665, dated 08 / 26 / 2025, pp. 168 / 234 5 / 64 treatment of endometriosis in a woman who needs it, comprising administering to said women an effective amount of an anti-netrin-1 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing an anti-netrin-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0017] The method involves the reduction of endometriosis lesions, especially uterine and / or uterine horn endometriosis lesions.

[0018] In an additional aspect, the aforementioned method comprises the reduction of endometriosis lesions, especially uterine and / or uterine horn endometriosis lesions, and the reduction of chronic pelvic pain relief or vaginal hyperalgesia relief.

[0019] In other respects, the aforementioned method also includes one or more of the following additional effects: - a reduction in colorectal hyperalgesia developed in endometriosis. - an improvement in bladder dysfunction developed in endometriosis. - a reduction in skin sensitivity to thermal stimuli, which is increased in endometriosis. - a reduction in the increased skin sensitivity to mechanical stimuli developed in endometriosis. - an improvement in overall well-being altered by endometriosis.

[0020] The percentage reduction or improvement of these effects disclosed above also applies here.

[0021] In another aspect, the invention relates to the use of a Petition 870250075665, dated 08 / 26 / 2025, pp. 169 / 234 6 / 64 anti-netrin-1 antibody or an antigen-binding fragment thereof, as disclosed herein, for the manufacture of a medicament to treat endometriosis and, preferably, endometriosis and pain, as disclosed herein. Detailed Description of the Invention

[0022] The antibody may be a polyclonal or monoclonal antibody that binds specifically to netrin-1 (NTN1) (anti-netrin-1 antibody or antibody that binds to netrin-1), especially to human netrin-1.

[0023] A polyclonal anti-netrin-1 antibody can, inter alia, be obtained by immunizing an animal, such as a rabbit, a mouse, and the like, with the aid of the selected amino acid sequence, collecting and then exhausting the antiserum obtained in, for example, an immunoadsorbent containing the receptor according to methods known per se to a person skilled in the art.

[0024] The amino acid sequence of netrin-1 is as described in SEQ ID NO: 1 and netrin-1 can be used in whole or in part to generate polyclonal or monoclonal antibodies.

[0025] Generally, monoclonal antibodies can be obtained according to the conventional method of lymphocyte fusion and hybridoma culture described by Kohler and Milstein (Nature, 1975, 256(5517): 495-7). Other methods for the preparation of monoclonal antibodies are also known (Harlow et al., ed., 1988, “Antibodies: a laboratory manual”). Monoclonal antibodies can be prepared by immunizing a mammal (e.g., a mouse, a rat, a rabbit, or even a human, similarly) and using the lymphocyte fusion technique that leads to hybridoma (Kohler and Milstein, 1975). There are alternative techniques to this usual technique. It is possible, for example, to produce monoclonal antibodies by expressing a nucleic acid cloned from a hybridoma. It is also possible to produce antibodies by Petition 870250075665, dated 08 / 26 / 2025, pp. 170 / 234 7 / 64 Phage display technique, introducing antibody cDNAs into vectors, which are typically filamentous phages displaying V gene libraries on the phage surface (e.g., fUSE5 for E. coli, Scott JK, Smith GP Science 1990; 249:386-390). Protocols for constructing these antibody libraries are described in JD Marks et al., J. Mol. Biol., 222 (1991), p. 581). The cDNA corresponding to the complete netrin-1 signal sequence (SEQ ID NO: 2) or a suitable fragment thereof can be used to produce monoclonal antibodies according to these methods.

[0026] The anti-netrin-1 monoclonal antibody (mAb) may be a murine, chimeric, humanized, or whole human monoclonal antibody. The fragment may be any type of mAb that substantially retains the ability of the whole antibody to bind to netrin-1; it may be, for example, a Fab or an F(ab')2. In particular, a monoclonal antibody is one disclosed in documents WO 2015 / 104360 or US 10,494,427, which documents are incorporated herein by reference, and which disclose useful murine, chimeric, and humanized monoclonal antibodies, and methods for the preparation of the various mAbs presented herein (e.g., HUM01-10 and similar). These are antibodies or fragments thereof that bind specifically to a netrin-1 epitope or to a polypeptide with the amino acid sequence SEQ ID NO: 3 or 33, or a variant thereof.

[0027] The antibodies useful in the invention can be defined by their CDRs. In particular, these CDRs are derived from the murine antibody 4C11 disclosed in WO 2015 / 104360 or US 10,494,427, an antibody that specifically binds to the polypeptide with the amino acid sequence SEQ ID NO: 3 or 33. Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a variable VH domain comprising: - an H-CDR1 with a sequence defined as SEQ ID NO: Petition 870250075665, dated 08 / 26 / 2025, pp. 171 / 234 8 / 64 5; - an H-CDR2 with a sequence defined as SEQ ID NO: 6; - an H-CDR3 with a sequence defined as SEQ ID NO: 7; a variable domain (VL) comprising: - an L-CDR1 with a sequence defined as SEQ ID NO: 8; - an L-CDR2 with a YAS sequence; - an L-CDR3 with a sequence defined as SEQ ID NO: 9; or a variable VH domain comprising: - an H-CDR1 with a sequence defined as SEQ ID NO: 28; - an H-CDR2 with a sequence defined as SEQ ID NO: 29; - an H-CDR3 with a sequence defined as SEQ ID NO: 30; a variable domain (VL) comprising: - an L-CDR1 with a sequence defined as SEQ ID NO: 31; - an L-CDR2 with a sequence defined as SEQ ID NO: 32; - an L-CDR3 with a sequence defined as SEQ ID NO: 9.

[0028] In a first series of embodiments, the antibody of the invention comprises an amino acid sequence of SEQ ID NO: 10, 11, Petition 870250075665, dated 08 / 26 / 2025, pp. 172 / 234 9 / 64 or 13. Typically, it comprises a VH of sequence SEQ ID NO: 10 and a VL of sequence SEQ ID NO: 11, or a heavy strand of sequence SEQ ID NO: 12 and a light strand of sequence SEQ ID NO: 13.

[0029] In a second series of embodiments, the antibody is chimeric. Preferably, it comprises a VH with sequence SEQ ID NO: 27 and a VL with sequence SEQ ID NO: 19.

[0030] In a third series of embodiments, the antibody is humanized. Preferably, it comprises an amino acid sequence selected from the SEQ ID NO: 14 to 18 (VL) group and / or the SEQ ID NO: 20 to 26 (VH) group. Typically, the humanized antibody comprises a VH with an amino acid sequence selected from the SEQ ID NO: 14 to 18 group and a VL with an amino acid sequence selected from the SEQ ID NO: 20 to 26 group.

[0031] The antibody preferably comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof, wherein the mAb or its fragment binds specifically to Netrin-1. The mAb may be a murine, chimeric, humanized, or fully human monoclonal antibody. The fragment may be any type of mAb that substantially retains the ability of the complete antibody to bind to Netrin-1; it may be, for example, a Fab or an F(ab')2.

[0032] The specific embodiments disclosed in this prior art document and which may be used herein are the following antibodies listed in Table 1. The first antibody listed is a chimeric 4C11 antibody, comprising murine VH and VL of the murine 4C11 antibody. The HUM00 listed in Table 1 corresponds to the grafting of murine 4C11 CDRs onto a human IgG1. The ten humanized mAbs HUM01 to HUM10 correspond to humanized mAbs derived from HUM00 with the same CDRs, but with specific modifications in the IgG FR regions. Petition 870250075665, dated 08 / 26 / 2025, pp. 173 / 234 10 / 64 human. HUM03 is also publicly known as NP137 and is currently in clinical trials. The human IgG1 CH antibody sequences are from Genbank AEL33691.1 modified R97K. The human IgG1 CL (Kappa) antibody sequences are from Genbank CAC20459.1. The other allotypes may also be used. The specific binding of all these mAbs, murine, chimeric and humanized HUM01-HUM10, Fab fragments and F(ab')2 fragments, to Netrin-1 and their ability to inhibit the binding of netrin-1 to its receptor UNC5B, are demonstrated in US 10,494,427 (Example 3).

[0033] In particular, these antibodies bind specifically to the polypeptide that has the amino acid sequence SEQ ID NO: 33. Table 1 Table 1 VH of SEQ ID NO: Constant heavy chain VL of SEQ ID NO: Constant light chain 4C11 10 Human IgG1 11 Human IgG1 HUM00 27 Human IgG1 19 Human IgG1 HUM01 20 Human IgG1 14 Human IgG1 HUM02 21 Human IgG1 15 Human IgG1 HUM03 22 Human IgG1 16 Human IgG1 HUM04 23 Human IgG1 17 Human IgG1 HUM05 24 Human IgG1 17 Human IgG1 HUM06 25 Human IgG1 16 Human IgG1 HUM07 26 Human IgG1 17 Human IgG1 HUM08 22 Human IgG1 17 Human IgG1 HUM09 25 Human IgG1 18 Human IgG1 HUM10 21 Human IgG1 16 Human IgG1

[0034] Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a pair of VH and VL sequences selected from the following pairs: SEQ ID NO: 27 and 19, SEQ ID NO: 20 and 14, SEQ ID NO: 21 and 15, SEQ ID NO: 22 and 16, SEQ ID NO: 23 and 17, SEQ ID NO: 24 and 17, SEQ ID NO: 25 and 16, SEQ ID NO: 26 and 17, SEQ ID NO: 22 and 17, SEQ ID NO: 25 and 18, SEQ ID NO: 21 and 16. More preferably, the antibody is a monoclonal antibody or a fragment of Petition 870250075665, dated 08 / 26 / 2025, pp. 174 / 234 11 / 64 antigen linkage of the same, comprising a pair of VH and VL sequences of SEQ ID NO: 22 and 16.

[0035] The anti-netrin-1 antibody may further comprise a constant heavy chain (CH) of human IgG1 and / or a constant light chain (CL) of human IgG1, in particular a constant human kappa domain.

[0036] In one embodiment, the human IgG1 CH sequences are sourced from Genbank AEL33691.1 modified R97K. The human IgG1 CL (Kappa) sequences are sourced from Genbank CAC20459.1. In one embodiment, the mAb is NP137 (AB_2811180 in The Antibody Registry) and comprises SEQ ID NO: 22 and 16 as VH and VL sequences, respectively, and the specific IgG1 CH and CL sequences.

[0037] The term “antigen-binding fragment” of an antibody (or “antibody-binding portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to bind specifically to netrin-1. An 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 aspect, the fragment comprises the VH and VL sequences of an antibody selected from HUM00 to HUM10.

[0038] Other antibodies, especially monoclonal antibodies or their antigen-binding fragments, developed against human netrin-1 or animal netrin-1, can be used as anti-netrin-1 antibodies, since netrin-1 is highly homologous across species. Examples include: Abcam antibodies ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, AF128.

[0039] In one embodiment, an antibody or the antigen-binding fragment thereof, with applications in a use or method of the present invention, is administered as a single dose, or an initial dose. Petition 870250075665, dated 08 / 26 / 2025, pp. 175 / 234 12 / 64 followed by the administration of a second or a plurality of subsequent doses of the antibody or the antigen-binding fragment thereof, wherein the subsequent doses are separated by at least one day; at least one week; at least two weeks; at least three weeks; at least four weeks; at least five weeks; at least six weeks; at least seven weeks; at least eight weeks; at least nine weeks; at least ten weeks; at least 12 weeks; or at least 14 weeks. The dose may vary depending on the age and weight of the individual to be administered, the target disease, conditions, route of administration, and the like. When the antibody of the present invention is used to treat pain in an adult patient, it is advantageous to administer it intravenously, subcutaneously, or intraperitoneally.A single dose or subsequent doses may comprise an amount of antibody or fragment thereof of at least 0.1 or 1 mg / kg of body weight, for example, approximately 1 mg / kg of body weight to approximately 100 mg / kg of body weight, in particular approximately 10 mg / kg of body weight to approximately 60 mg / kg of body weight in the individual. 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. Definitions and Other Embodiments, Variants and Alternatives of the Invention

[0040] Throughout this application, the term “comprising” shall be interpreted as encompassing all the features specifically mentioned, as well as those that are optional, additional, and unspecified. As used herein, the use of the term “comprising” also describes the embodiment in which no other feature besides the features specifically mentioned is present (i.e., “consisting of”, “contributing to”). Petition 870250075665, dated 08 / 26 / 2025, pp. 176 / 234 13 / 64

[0041] In the context of the invention, the term “treat” or “treatment”, as used herein, means to obtain a reduction or disappearance of the lesions and, preferably, a reduction or relief of pain related to endometriosis and / or a comorbidity associated with endometriosis, as disclosed herein.

[0042] By “therapeutically effective amount” of the antibody of the invention, it is meant an amount sufficient to treat the said pain, with a reasonable benefit / risk ratio applicable to any medical treatment. It should be understood, however, that the total daily use of the antibody will be decided by the attending physician, within the scope of good medical judgment.The specific therapeutically effective dose level for any given patient will depend on a variety of factors, including the severity of endometriosis, such as the number and extent of lesions, and the degree of pain and / or comorbidities; the activity of the specific antibody used; the specific composition used; the age, body weight, general health, and diet of the women; the timing of administration, the route of administration, and the excretion rate of the specific antibody used; the duration of treatment; drugs used in combination with or concomitantly with the specific antibody used; and similar factors well known in the medical field.

[0043] “Pharmaceutically” or “pharmaceutically acceptable” refers to entities and molecular compositions that do not produce adverse, allergic or other adverse reactions when administered to a mammal, especially a human being, as appropriate. A pharmaceutically acceptable vehicle (or carrier or excipient) refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any kind.

[0044] The form of the pharmaceutical compositions, including the antibody of the invention, and the route of administration naturally depend on Petition 870250075665, dated 08 / 26 / 2025, pp. 177 / 234 14 / 64 condition to be treated, the severity of the disease, the patient's age, weight and sex, etc.

[0045] The antibody of the invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. In one specific embodiment, the antibody of the invention is administered intravenously.

[0046] In particular, pharmaceutical compositions that include the antibody of the invention may contain pharmaceutically acceptable vehicles for an injectable formulation. These may be, in particular, isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like, or mixtures of these salts), or dry compositions, especially lyophilized ones, which, after the addition, as the case may be, of sterile water or physiological saline, allow the constitution of injectable solutions.

[0047] Suitable pharmaceutical forms for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid, so as to allow easy administration by syringe. It must be stable under manufacturing and storage conditions and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0048] The vehicle or carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Adequate fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the particle size required in the case of dispersion, and by the use of surfactants, stabilizing agents, cryoprotectants or antioxidants. Prevention of the action of Petition 870250075665, dated 08 / 26 / 2025, pp. 178 / 234 15 / 64 microorganisms can be controlled by antibacterial and antifungal agents. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0049] Sterile injectable solutions are prepared by incorporating the active compounds in the required quantity into the appropriate solvent with several of the other ingredients listed above, as needed, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization techniques, which produce a powder of the active ingredient plus any desired additional ingredient from a previously sterile filtered solution.

[0050] After formulation, the solutions will be administered in a manner consistent with the formulation dosage and in a therapeutically effective quantity. The formulations are easily administered in various pharmaceutical forms, such as the injectable solutions described above, but drug-release capsules and similar forms can also be used.

[0051] For parenteral administration in aqueous solution, for example, the solution should be adequately buffered, if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose solution. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be employed will be known to those skilled in the art, in light of this disclosure. For example, a dosage can be dissolved in 1 mL of isotonic solution of Petition 870250075665, dated 08 / 26 / 2025, pp. 179 / 234 16 / 64 NaCl is added to 1000 mL of hypodermoclysis fluid or injected into the proposed infusion site (see, for example, “Remington's Pharmaceutical Sciences”, 15th edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the individual being treated. The person responsible for administration will determine, in any case, the appropriate dose for the individual. Table 2: Description of the sequences: SEQ ID NO: Description Sequence 1 Amino acid (aa) sequence of netrin-1 with signal peptide in bold and linear epitope mapping in bold and underlined MMRA VWEALAALAAVA CLVGAVRG G PG LSMFAGQAA QPDPCSDENGHPRRCIPDFVNAAFGKDVRVSSTCGRPP ARYCVVSERGEERLRSCHLCNASDPKKAHPPAFLTDLN NPHNLTCWQSENYLQFPHNVTLTLSLGKKFEVTYVSLQF CSPRPESMAIYKSMDYGRTWVPFQFYSTQCRKMYNRP HRAPITKQNEQEAVCTDSHTDMRPLSGGLIAFSTLDGRP SAHDFDNSPVLQDWVTATDIRVAFSRLHTFGDENEDDS ELARDSYFYAVSDLQVGGRCKCNGHAARCVRDRDDSL VCDCRHNTAGPECDRCKPFHYDRPWQRATAREANECV acncnlharrcrfnmelyklsgrksggvclncrhnta GRHCHYCKEGYYRDMGKPITHRKACKACDCHPVGAAG KTCNQTTGQCPCKDGVTGITCNRCAKGYQQSRSPIAPCI KIPVAPPTTAASSVEEPEDCDSYCKASKGKLKINMKKYC KKDYAVQIHILKADKAGDWWKFTVNIISVYKQGTSRIRRG DQSLWIRSRDIACKCPKIKPLKKYLLLGNAEDSPDQSGIV ADKSSLVIQWRDTWARRLRKFQQREKKGKCKKA 2 Nucleic acid sequence of netrin-1.ATGATGCGCGCAGTGTGGGAGGCGCTGGCGGCGCTG GCGGCGGTGGCGTGCCTGGTGGGCGCGGTGCGCGG CGGGCCCGGGCTCAGCATGTTCGCGGGCCAGGCGG CGCAGCCCGATCCCTGCTCGGACGAGAACGGCCACC CGCGCCGCTGCATCCCGGACTTTGTCAATGCGGCCTT CGGCAAGGACGTGCGCGTGTCCAGCACCTGCGGCCG GCCCCCGGCGCGCTACTGCGTGGTGAGCGAGCGCG GCGAGGAGCGGCTGCGCTCGTGCCACCTCTGCAACG CGTCCGACCCCAAGAAGGCGCACCCGCCCGCCTTCC TCACCGACCTCAACAACCCGCACAACCTGACGTGCTG GCAGTCCGAGAACTACCTGCAGTTCCCGCACAACGTC ACGCTCACACTGTCCCTCGGCAAGAAGTTCGAAGTGA CCTACGTGAGCCTGCAGTTCTGCTCGCCGCGGCCCG AGTCCATGGCCATCTACAAGTCCATGGACTACGGGCG CACGTGGGTGCCCTTCCAGTTCTACTCCACGCAGTGC CGCAAGATGTACAACCGGCCGCACCGCGCGCCCATC ACCAAGCAGAACGAGCAGGAGGCCGTGTGCACCGAC TCGCACACCGACATGCGCCCGCTCTCGGGCGGCCTC ATCGCCTTCAGCACGCTGGACGGGCGGCCCTCGGCG CACGACTTCGACAACTCGCCCGTGCTGCAGGACTGG GTCACGGCCACAGACATCCGCGTGGCCTTCAGCCGC CTGCACACGTTCGGCGACGAGAACGAGGACGACTCG GAGCTGGCGCGCGACTCGTACTTCTACGCGGTGTCC GACCTGCAGGTGGGCGGCCGGTGCAAGTGCAACGGC CACGCGGCCCGCTGCGTGCGCGACCGCGACGACAG. Petição 870250075665, de 26 / 08 / 2025, pág. 180 / 234 17 / 64 SEQ ID NO: Descrição Sequência CCTGGTGTGCGACTGCAGGCACAACACGGCCGGCCC GGAGTGCGACCGCTGCAAGCCCTTCCACTACGACCG GCCCTGGCAGCGCGCCACAGCCCGCGAAGCCAACGA GTGCGTGGCCTGTAACTGCAACCTGCATGCCCGGCG CTGCCGCTTCAACATGGAGCTCTACAAGCTTTCGGGG CGCAAGAGCGGAGGTGTCTGCCTCAACTGTCGCCAC AACACCGCCGGCCGCCACTGCCATTACTGCAAGGAG GGCTACTACCGCGACATGGGCAAGCCCATCACCCAC CGGAAGGCCTGCAAAGCCTGTGATTGCCACCCTGTG GGTGCTGCTGGCAAAACCTGCAACCAAACCACCGGC CAGTGTCCCTGCAAGGACGGCGTGACGGGTATCACC TGCAACCGCTGCGCCAAAGGCTACCAGCAGAGCCGC TCTCCCATCGCCCCCTGCATAAAGATCCCTGTAGCGC CGCCGACGACTGCAGCCAGCAGCGTGGAGGAGCCTG AAGACTGCGATTCCTACTGCAAGGCCTCCAAGGGGAA GCTGAAGATTAACATGAAAAAGTACTGCAAGAAGGAC TATGCCGTCCAGATCCACATCCTGAAGGCGGACAAGG CGGGGGACTGGTGGAAGTTCACGGTGAACATCATCTC CGTGTATAAGCAGGGCACGAGCCGCATCCGCCGCGG TGACCAGAGCCTGTGGATCCGCTCGCGGGACATCGC CTGCAAGTGTCCCAAAATCAAGCCCCTCAAGAAGTAC CTGCTGCTGGGCAACGCGGAGGACTCTCCGGACCAG AGCGGCATCGTGGCCGATAAAAGCAGCCTGGTGATC CAGTGGCGGGACACGTGGGCGCGGCGGCTGCGCAA GTTCCAGCAGCGTGAGAAGAAGGGCAAGTGCAAGAA GGCCTAGCG 3 Sequência epitópica do aade netrina-1. VACNCNLHARRCRFNMELYKLSGRKSGGVCLNCRHNT AGRHCH 4 Sequência of cDNA epitópica of netrina-1. GTGGCCTGTAACTGCAACCTGCATGCCCGGCGCTGC CGCTTCAACATGGAGCTCTACAAGCTTTCGGGGCGCA AGAGCGGAGGTGTCTGCCTCAACTGTCGCCACAACAC CGCCGGCCGCCACTGCCAT 5 seq. de aa de CDR1-H (IMGT) GYTFTSYN 6 seq. de aa de CDR2-H (IMGT) IYPGNGDT 7 seq. de aa de CDR3-H (IMGT) ARGGTGFAY 8 seq. de aa de CDR1-L (IMGT) QSVSND - seq. de aa de CDR2-L (IMGT) YAS 9 seq. de aa de CDR3-L (IMGT e Kabat) QQDYSSPWT 10 seq. de aa de VH 4C11 de camundongo QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWV KQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSS STAYMQLSSLTSEDSAVYFCARGGTGFAYWGQGTLVTV SA 11 seq. de aa de VL 4C11 de camundongo SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQK PGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTV QAEDLAVYFCQQDYSSPWTFGGGTKLEIK 12 Seq. de aa completa QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWV Petition: 870250075665, on August 26, 2025, p. 181 / 234 18 / 64 SEQ ID NO: Description Sequence of 4C11 (VH + CH of IgG1 of camundongo) KQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSS STAYMQLSSLTSEDSAVYFCARGGTGFAYWGQGTLVTV TVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRP SETVTCCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVS SVFIFPPKPKDVLTITLTPKVTCVVVDISKDDDPEVQFSWFV DDVVHTAQTQPREEQFNSTFRSVSELPIHQDWLNGK EFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQ PIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLH NHHTEKSLSHSPGK 13 Seq. of aa complete of 4C11 (VL + CL Kappa de camundongo) SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQK PGQSPKLLIYASNRYTGVPDRFTGSGYGTDFTFTISTV QAEDLAVYFCQDYSSPWTFGGGTDAPPKSIKSIKSITV FPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNEC 14 Aa sequence of VL from the humanized variant of 4C11 EIVMTQSPATLSVASSCVASCVASCVAQQSQTLSW KPGKAPKLLIYASNRYTGIPPRFSGSGYGTDFTLTINNIE SEDAAYYFCQQDYSSPWTFGQG 15 Aa sequence of VL from the humanized variant of 4C11DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWFQQ RPGQSPRRLIYYASNRYTGVPSRFSGSGSGTDFTFTISS LEAEDAATYYCQQDYSSPWTFGQG 16 VL sequence from humanized variant of 4C11 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQ KPGQAPRLLIYYASNRYTGIPPRFSGSGYGTDFTLTINNI ESEDAAYYFCQQDYSSPWTFGQG 17 Sequence of VL aa from humanized variant of 4C11 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYLQK PGQSPQLLIYYASNRYTGVPSRFSGSGSGTDFTFTISSL EAEDAATYYCQQDYSSPWTFGQG 18 Sequência de aa de VL da variante humanizada de 4C11 DIVMTQTPLSLPVTPGEPASISCKASQSVSNDVAWYQQK PGQAPRLLIYYASNRYTGIPPRFSGSGYGTDFTLTINNIE SEDAAYYFCQQDYSSPWTFGQG 19 Sequência de aa de VL da variante humanizada de 4C11 EIVMTQSPATLSVSPGERATLSCRASQSVSNDVAWYQQ KPGQAPRLLIYYASNRYTGIPARFSGSGSGTEFTLTISSL QSEDFAVYYCQQDYSSPWTFGQG 20 Sequência de aa de VH da variante humanizada de 4C11 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWV RQATGQGLEWMGAIYPGNGDTSYNQKFKGRVTITADKS TSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG 21 Sequence of VH aa from humanized variant of 4C11QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWIRQ PPGKGLEWIGAIYPGNGDTSYNQKFKGRVTITADKSTST AYMELSSLRSEDTAVYYCARGGTGFAYWGQG 22 Sequência de aa de VH da variante humanizada de 4C11 QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWVR QATGQGLEWMGAIYPGNGDTSYNQKFKGRLTISKDTSK NQVVLTMTNMDPVDTATYYCARGGTGFAYWGQG 23 Sequência de aa de VH da variante humanizada de 4C11 EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYNMHWVR QATGQGLEWMGAIYPGNGDTSYNQKFKGRFTISRDDSK NTAYLQMNSLKTEDTAVYYCARGGTGFAYWGQG 24 Sequência de aa de VH da variante humanizada de 4C11 QVQLQESGPGLVKPSQTLSLTCTVSGYTFTSYNMHWVR QAPGQGLEWMGAIYPGNGDTSYNQKFKGRVTISVDTSK NQFSLKLSSVTAADTAVYYCARGGTGFAYWGQG 25 Sequência de aa de QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWV Petition 870250075665, 08 / 26 / 2025, pág. 182 / 234 19 / 64 SEQ ID NO: Description VH sequence from humanized variant of 4C11 rqatgqglewmgaiypgngdtsynqkfkgrvtitadks TSTAYMELSSLRSEDTAVYYCARGGTGFAYWGQG 26 VH sequence from humanized variant of 4C11 QVQLQQSGPGLVKPSQTLSLTCAISGYTFTSYNMHWVR qatgqglewmgaiypgngdtsynqkfkgrvtitadkst STAYMELSSLRSEDTAVYYCARGGTGFAYWGQG 27 VH aa sequence from humanized variant of 4C11 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWV rqapgqglewmgaiypgngdtsyaqkfkgrvtmtrdt STSTVYMELSSLRSEDTAVYYCARGGTGFAYWGQ 28 seq. de aa de CDR1-H (Kabat) SYNMH 29 seq. de aa de CDR2-H (Kabat) AIYPGNGDTSYNQKFKG 30 seq. de aa de CDR3-H (Kabat) GGTGFAY 31 seq. de aa de CDR1-L (Kabat) KASQSVSNDVA 32 seq. de aa de CDR2-L (Kabat) YASNRYT 33 seq. epitópica do aa de netrin-1. ARRCRFNMELYKLSGRKSGGVC 34 seq. de cDNA epitópico de netrin-1. GCCCGGCGCTGCCGCTTCAACATGGAGCTCTACAAG CTTTCGGGGCGCAAGAGCGGAGGTGTCTGC

[0052] The CDRs under IMGT are removed in black on the table 1, when appropriate. Definitions and other embodiments, variants and alternatives of the INVENTION

[0053] As used herein, a sequence “at least 85% identical to a reference sequence” is a sequence that has, throughout its length, 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% sequence identity with the entire length of the reference sequence.

[0054] A percentage of “sequence identity” can be determined by comparing the two sequences, ideally aligned in a comparison window, in which the portion of the polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is Petition 870250075665, dated 08 / 26 / 2025, pp. 183 / 234 20 / 64 is calculated by determining the number of positions where the identical amino acid residue occurs in both sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate the sequence identity percentage. The ideal alignment of sequences for comparison is performed by global pairwise alignment, for example, using the Needleman and Wunsch algorithm (1970) J. Mol. Biol. 48: 443. The sequence identity percentage can be easily determined, for example, using the Needle program, with the BLOSUM62 matrix and the following parameters: gap opening = 10, gap length = 0.5.

[0055] In the context of the invention, a “conservative amino acid substitution” is one 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). In general, a conservative amino acid substitution does not substantially alter the functional properties of a protein. Examples of amino acid groups that have side chains with 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 conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0056] An “antibody” can be a natural antibody or Petition 870250075665, dated 08 / 26 / 2025, pp. 184 / 234 The conventional 21 / 64 antibody isotype, in which two heavy chains are linked together by disulfide bridges, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda and kappa. There are five main classes of heavy chains (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light (VL) and heavy (VH) chains determine recognition binding and antigen specificity.The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of a light chain and a heavy chain. Antibody specificity resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are composed of residues that are primarily from hypervariable regions or complementarity determinants (CDRs). Occasionally, residues from non-hypervariable regions or structure (FR) regions influence the overall structure of the domain and, consequently, the binding site.

[0057] “Complementarity-Determining Regions” or “CDRs” refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H and CDR3-H, respectively. An antigen-binding site Petition 870250075665, dated 08 / 26 / 2025, pp. 185 / 234 22 / 64 of conventional antibody, therefore, includes six CDRs, comprising the set of CDRs from each V region of the heavy and light chain.

[0058] “Structure Regions” (FRs) refer to amino acid sequences interposed between CDRs, that is, to the portions of the variable regions of the light and heavy chains of immunoglobulin that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H and FR4-H, respectively.

[0059] As used herein, a “human structure region” is a structure region that is substantially identical (about 85% or more, in particular 90%, 95%, 97%, 99% or 100%) to the structure region of a naturally occurring human antibody.

[0060] In the context of the invention, the definition of CDR / FR in an immunoglobulin light or heavy chain must be determined based on the Kabat or IMGT definitions.

[0061] Residues in variable antibody domains are conventionally numbered according to a system developed by Kabat et al. This system is established in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al.”). This numbering system is used in this descriptive report. Kabat residue designations do not always directly correspond to the linear numbering of amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer amino acids or additional amino acids than in the strict Kabat numbering, corresponding to a shortening or insertion into a structural component, either a structural framework or a complementarity-determining region (CDR), of the basic structure of the antibody. Petition 870250075665, dated 08 / 26 / 2025, pp. 186 / 234 23 / 64 variable domain. The correct Kabat numbering of residues can be determined for a given antibody by aligning homology residues in the antibody sequence with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (HCDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3), according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3), according to the Kabat numbering system. (http: / / www.bioinf.org.uk / abs / #cdrdef)

[0062] In the context of the invention, the amino acid residues of the antibody of the invention can be numbered according to the IMGT numbering system. The unique IMGT numbering system was defined to compare variable domains, regardless of antigen receptor, chain type, or species (Lefranc M.-P., “Unique database numbering system for immunogenetic analysis” Immunology Today, 18, 509 (1997); Lefranc M.-P., “The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains” The Immunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G., “Unique IMGT numbering for variable domains of the immunoglobulin receptor and T and V-like domains of the Ig superfamily” Dev. Comp. Immunol., 27, 55-77). (2003).).In the unique IMGT numbering system, conserved amino acids always have the same position, for example, cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine or tryptophan 118. The unique IMGT numbering system provides a standardized delineation of the structure regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and the complementarity-determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the top of the... Petition 870250075665, dated 08 / 26 / 2025, pp. 187 / 234 24 / 64 loop, in the following order: 111, 112, 110, 113, 109, 114, etc. If the length of CDR3-IMGT is greater than 13 amino acids, additional positions will be created between positions 111 and 112 at the top of the CDR3-IMGT loop in the following order: 112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc. (http: / / www.imgt.org / IMGTScientificChart / Nomenclature / IMGTFRCDRdefinition.html).

[0063] As used herein, the term “antibody” denotes conventional antibodies and fragments thereof, as well as single-domain antibodies and fragments thereof, in particular single-domain variable heavy chain antibodies and chimeric, humanized, bispecific or multispecific antibodies.

[0064] As used herein, antibody or immunoglobulin also includes “single-domain antibodies,” which have been described more recently and are antibodies whose complementary determinant regions are part of a single-domain polypeptide. Examples of single-domain antibodies include heavy-chain antibodies, naturally occurring light-chain-deficient antibodies, single-domain antibodies derived from conventional four-chain antibodies, and engineered single-domain antibodies. Single-domain antibodies can be derived from any species, including but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. Single-domain antibodies can be naturally occurring single-domain antibodies, known as light-chain-deficient heavy-chain antibodies.In particular, Camelidae species, for example, camel, dromedary, llama, alpaca, and guanaco, produce heavy chain antibodies that are naturally devoid of light chains. Camelid heavy chain antibodies also lack the CH1 domain.

[0065] The variable heavy chain of these domain antibodies Petition 870250075665, dated 08 / 26 / 2025, pages 188 / 234 Unique 25 / 64, devoid of light chains, is known in the art as “VHH” or “nanobody”. Similar to conventional VH domains, VHHs contain four RFs and three CDRs. Nanobodies offer advantages over conventional antibodies: they are about ten times smaller than IgG molecules and, as a consequence, appropriately folded functional nanobodies can be produced by in vitro expression with high yield. Furthermore, nanobodies are very stable and resistant to protease action. The properties and production of nanobodies were reviewed by Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22.

[0066] The term “monoclonal antibody” or “mAb”, as used herein, refers to an antibody molecule with a single amino acid composition that is directed against a specific antigen, and should not be interpreted as requiring the production of the antibody by any specific method. A monoclonal antibody can be produced by a single clone of B cells or hybridoma, but it can also be recombinant, i.e., produced by protein engineering.

[0067] “Fragments” of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single-domain antibody, such as a heavy chain or VHH antibody.

[0068] The term “Fab” denotes an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, in which approximately half of the N-terminal side of the H chain and the entire L chain, among the fragments obtained by treating IgG with a protease, papain, are Petition 870250075665, dated 08 / 26 / 2025, pp. 189 / 234 26 / 64 linked by a disulfide bond.

[0069] The term “F(ab')2” refers to an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity that is slightly higher than Fab linked via a disulfide bond in the hinge region between fragments obtained by treating IgG with a protease, pepsin.

[0070] A single-chain Fv polypeptide (“scFv”) is a covalently linked VH::VL heterodimer, which is generally expressed from a gene fusion that includes genes encoding VH and VL, linked by a peptide-coding linker. The human scFv fragment of the invention includes CDRs that are maintained in an appropriate conformation, in particular by the use of gene recombination techniques. Divalent and multivalent antibody fragments can form spontaneously by the association of monovalent scFvs or can be generated by the coupling of monovalent scFvs by a peptide linker, such as the divalent sc(Fv)2.

[0071] “dsFv” is a VH::VL heterodimer stabilized by a disulfide bond.

[0072] “(dsFv)2” denotes two dsFv coupled by a peptide ligand.

[0073] The term “bispecific antibody” or “BsAb” refers to an antibody that combines the antigen-binding sites of two antibodies into a single molecule. Thus, BsAbs are able to bind to two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described, for example, in EP 2 050 764 A1.

[0074] The term “multispecific antibody” denotes an antibody that combines the antigen-binding sites of two or more antibodies into one. Petition 870250075665, dated 08 / 26 / 2025, pp. 190 / 234 27 / 64 single molecule.

[0075] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, these fragments comprising a variable heavy chain (VH) domain connected to a variable light chain (VL) domain on the same polypeptide chain (VH-VL). By using a ligand that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites.

[0076] In a particular embodiment, the epitope-linking fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diacorpos and VHH.

[0077] A “chimeric antibody,” as used herein, is an antibody in which the constant region, or a portion thereof, is altered, substituted, or exchanged, so that the variable region is linked to a constant region of a different species or belonging to another class or subclass of antibodies. “Chimeric antibody” also refers to an antibody in which the variable region, or a portion thereof, is altered, substituted, or exchanged, so that the constant region is linked to a variable region of a different species or belonging to another class or subclass of antibodies.

[0078] The term “humanized antibody” refers to an antibody that is initially wholly or partially of non-human origin and has been modified to replace certain amino acids, particularly in the structural regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most often human CH and CL domains. In one embodiment, a humanized antibody possesses constant domains of human origin. As used herein, the term “humanized antibody” refers to a chimeric antibody containing a minimal sequence derived from Petition 870250075665, dated 08 / 26 / 2025, pp. 191 / 234 28 / 64 non-human immunoglobulin, for example, CDRs.

[0079] The term “antibody” is used to encompass all these types of antibodies, fragments or combinations thereof.

[0080] The goal of humanization is to reduce the immunogenicity of a xenogeneic antibody, such as a murine antibody, for introduction into a human, while maintaining the antibody's affinity and antigen-binding specificity. Humanized antibodies, or antibodies adapted for non-rejection by other mammals, can be produced using various technologies, such as resurfacing and CDR grafting. As used in this work, resurfacing technology uses a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surfaces of the antibody's variable regions to resemble the surfaces of known antibodies from the target host.

[0081] Antibodies can be humanized using a variety of other techniques, including CDR grafting (EP 0 239 400; WO 91 / 09967; US Patents Nos. 5,530,101 and 5,585,089), coating or resurfacing (EP 0 592 106; EP 0 519 596; Padlan (1991) Molecular Immunology 28(4 / 5):489-498; Studnicka et al. (1994) Protein Engineering 7(6):805-814; Roguska et al. Human antibodies can be produced by a variety of methods known in the art, including phage display methods. See also US Patents Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and international patent application WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741.

[0082] As used in this document, the term “specificity” refers to the ability of an antibody to bind detectably to an epitope present on an antigen, such as netrin-1, Petition 870250075665, dated 08 / 26 / 2025, pp. 192 / 234 29 / 64 while exhibiting relatively little detectable reactivity with non-netrin-1 proteins or structures (such as other proteins present in cancer cells or other cell types). Specificity can be determined relatively by binding or competitive binding assays, using, for example, Biacore instruments, as described elsewhere in this document. Specificity can be demonstrated by, for example, an affinity / avidity ratio of approximately 10:1, approximately 20:1, approximately 50:1, approximately 100:1, 10,000:1 or higher in binding to the specific antigen versus non-specific binding to other irrelevant molecules (in this case, the specific antigen is netrin-1).

[0083] The term “affinity,” as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [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 the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), whose references are incorporated herein by reference. A preferred and well-known standard method in the art for determining mAbs affinity is the use of Biacore instruments.

[0084] The present invention will now be described in more detail using the following non-limiting experiments relating to the Figures. Brief Description of the Figures

[0085] Figure 1: Schematic diagram showing the timeline for different interventions performed in this study. Petition 870250075665, dated 08 / 26 / 2025, pp. 193 / 234 30 / 64

[0086] Figure 1A) Mice were ovariectomized one week (-1 week) prior to surgical induction of uterine horn fragments (Endo) or suture only (Sham) on day 0 (d0). All mice were injected weekly with intraperitoneal (ip) estradiol benzoate (vertical red marks) to maintain stable circulating estrogen levels throughout the study. Treatment began 4 weeks after surgical induction, with groups consisting of Endo mice treated with NP137 (Endo + NP137 (N = 15)) or control antibody NP001 (Endo + Ab control (N = 16)) and Sham mice treated with Ab control NP001 (Sham + Ab control (N = 16)). Biweekly ip treatment was maintained throughout the duration of the study. In vivo pain assessment studies, comparing Sham (control Ab) and Endo (control Ab and NP137), were performed between weeks 8 and 10 after surgical induction (shaded box).After the final behavioral assessment, tissue was collected from all mice (diamonds). The total number of mice used for this study was N = 16 Sham (control Ab), N = 16 Endo (control Ab), and N = 15 Endo (NP137).

[0087] Figure 1B) Mice were ovariectomized one week (-1 week) prior to inoculation of uterine horn fragments (Endo) or saline solution only (Sham) on day 0 (d0). All mice were injected weekly with intraperitoneal (ip) estradiol benzoate (vertical markers) to maintain stable circulating estrogen levels throughout the study. Treatment began 4 weeks after surgical induction, with groups consisting of Endo mice treated with NP137 (Endo + NP137) or control antibody NP001 (Endo + Ab control (N = 8)) and Sham mice treated with Ab control NP001 (Sham + Ab control (N = 4)). Biweekly ip treatment was maintained throughout the duration of the study. In vivo pain assessment studies comparing Sham (Ab control) and Endo (Ab control and NP137) were performed between 8 and 10 weeks after surgical induction (shaded box). After the Petition 870250075665, dated 08 / 26 / 2025, pages 194 / 234 31 / 64 Behavioral assessment using the hot plate test, tissue was collected from all mice (diamonds). The total number of mice used was N = 4 Sham (Ab control), N = 8 Endo (Ab control) and N = 7 Endo (NP137).

[0088] Figure 2: Treatment with NP137 reduces the size of the endometriosis lesion.

[0089] Figure 2A1) Representative images of the reproductive tract 10 weeks after induction surgery for a Sham mouse treated with Ab control (left box, sutures only in the uterine horns and next to the uterus (arrows and arrowheads indicate positioning)), Endo mouse (endometrial fragments attached to the uterine horns and next to the uterus (arrows indicate positioning)) treated with Ab control (middle box) or NP137 (right box).

[0090] Figure 2A2) Representative images of dissected and cleaned endometriosis lesions, collected from the uterus and uterine horns of Endo mice treated with Ab control (4 mice, box on the left) and NP137 (3 mice, box on the right). The measuring bars represent 5 mm.

[0091] Figure 2B) Pooled data show a significant reduction in mean lesion weight 10 weeks after Endo induction following chronic treatment with NP137 compared to the Ab control.

[0092] Figure 2B1) Grouped data displayed as the average weight of all lesions per animal (g).

[0093] Figure 2B2) Grouped data displayed as the average weight of lesions located in the uterus (g).

[0094] Figure 2B3) Grouped data displayed as the average weight of lesions located in the uterine horns (g).

[0095] Figure 2C) Pooled data show a significant reduction in the mean maximum lesion diameter 10 weeks after induction of Petition 870250075665, dated 08 / 26 / 2025, pp. 195 / 234 32 / 64 Endoscopic lesions after chronic treatment with NP137 compared to the Ab control. Data represent the mean ± SEM. NS, P > 0.05, **P < 0.01, ***P < 0.001, non-parametric Mann-Whitney test for 2 groups with unequal variances of N = 9 to 11 mice, en = 29 to 35 lesions per treatment group.

[0096] Figure 2C1) Grouped data displayed as the mean maximum lesion diameter of all lesions per animal (mm).

[0097] Figure 2C2) Grouped data displayed as the mean maximum lesion diameter of lesions located in the uterus (mm).

[0098] Figure 2C3) Grouped data displayed as the mean maximum lesion diameter of lesions located in the uterine horns (mm).

[0099] Figure 3: Treatment with NP137 reduces vaginal hyperalgesia developed in mice with endometriosis.

[00100] Figure 3A) Pooled data showing elevated visceromotor responses (VMR) to vaginal distension (VD) in Endo mice treated with Ab control compared to Sham mice treated with Ab control from 20 mm Hg - 70 mm Hg. The elevated response in Endo mice was significantly reduced at non-noxious distension pressures in mice treated with NP137. Λ represents Ab control Sham vs. Ab control Endo and * represents Ab control Endo vs. Endo NP137. Data represent mean ± SEM with * or Λp < 0.05, ** or Λp < 0.01, **** or Λp < 0.0001, generalized estimating equations followed by LSD post hoc test.

[00101] Figures 3B-D) Pooled VD data show that the elevated responses seen in Ab-treated Endo mice compared to Ab-treated Sham mice are normalized by NP137 treatment. Data represent mean ± SEM with NS = not significant, *P < 0.05 and **P < 0.01, an ordinary one-way ANOVA with Sidak's post-hoc multiple comparison test for Petition 870250075665, dated 08 / 26 / 2025, pp. 196 / 234 33 / 64 groups of equal variances, with N = 12-14 mice per treatment group. Each point represents the AUC of an individual animal.

[00102] Figure 3B) Pooled data expressed as the total area under the VMR curve for all distending pressures (20-80 mm Hg).

[00103] Figure 3C) Pooled data expressed as the total area under the VMR curve for low distending pressures (20-40 mm Hg).

[00104] Figure 3D) Pooled data expressed as the total area under the VMR curve for high distending pressures (60-80 mm Hg).

[00105] Figure 4: Treatment with NP137 reduces colonic hyperalgesia developed in mice with endometriosis.

[00106] Figure 4A) Pooled data showing increased visceromotor responses (VMR) to colorectal distension (CRD) in Endo mice (yellow symbols) compared to Sham mice when treated with Ab control (blue symbols). The elevated response to CRD in Endo mice was significantly reduced in mice treated with NP137 (green symbols), from 20 mm Hg to 60 mm Hg. Λ represents Ab control Sham vs Ab control Endo and * represents Ab control Endo vs Endo NP137. Data represent mean ± SEM with * or Λρ < 0.05, *** or λρ < 0.001, **** or λρ < 0.0001, generalized estimating equations followed by the LSD post hoc test.

[00107] Figures 4 BD) Pooled CRD data show that the elevated responses seen in Ab-treated Endo mice compared to Ab-treated Sham mice are normalized by NP137 treatment. Data represent mean ± SEM with NS = not significant, **P < 0.01, **P < 0.001 and ****P < 0.0001, Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, from N = 12 - 14 mice per treatment group. Each point represents the AUC of an individual animal. Petition 870250075665, dated 08 / 26 / 2025, pp. 197 / 234 34 / 64

[00108] Figure 4B) Pooled data expressed as the total area under the VMR curve for all distending pressures (20-80 mm Hg).

[00109] Figure 4C) Pooled data expressed as the total area under the VMR curve for low distending pressures (20-40 mm Hg).

[00110] Figure 4D) Pooled data expressed as the total area under the VMR curve for high distending pressures (50-80 mm Hg).

[00111] Figure 5: Treatment with NP137 reverses bladder dysfunction developed in mice with endometriosis.

[00112] Figure 5A) Pooled data of total urine spot count in Sham mice treated with Ab control, Endo mice treated with Ab control and Endo mice treated with NP137 showing that the significant increase in total spot count produced by Endo mice was normalized with NP137 treatment.

[00113] Figure 5B) Pooled data showing voiding patterns (total urine blots) separated into small (100-1,000 dpi), medium (1,001-100,000 dpi) or large (>100,001 dpi) urine blots in Sham mice treated with Ab control (blue symbols), Endo mice treated with Ab control (yellow symbols) and Endo mice treated with NP137 (green symbols). The data show that the significant increase in small blots produced by Endo mice is normalized with NP137 treatment. Data represent mean ± SEM with NS = not significant, *P < 0.05 and **P < 0.01, ordinary one-way ANOVA with Tukey's post-hoc multiple comparison test for groups of equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, with N = 13 - 16 mice per treatment group. Each point represents an individual animal. Petition 870250075665, dated 08 / 26 / 2025, pp. 198 / 234 35 / 64

[00114] Figure 6: Treatment with NP137 reverses mechanical and thermal cutaneous hypersensitivity developed in mice with endometriosis.

[00115] Figure 6A) Pooled data showing the mechanical paw withdrawal threshold induced by stimulation of the von Frey electronic filament (EvF) of the hind paw (g) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137. The data show that the greater sensitivity to mechanical stimuli exhibited by Endo mice, compared to Sham mice treated with Ab control, is normalized with NP137 treatment.

[00116] Figure 6B) Pooled data showing the reaction latency to the heating plate (sec) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137. The data show that the increased sensitivity to noxious thermal stimuli exhibited by Endo mice compared to Sham mice treated with Ab control is absent in Endo mice treated with NP137. Data represent mean ± SEM with NS = not significant, *P < 0.05, **P < 0.01 and ****P < 0.0001, ordinary one-way ANOVA with Tukey's post-hoc multiple comparison test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, of N = 14 - 16 mice per treatment group. Each point represents an individual animal.

[00117] Figure 7: Treatment with NP137 improves signs of reduced overall well-being exhibited in nest building in mice with endometriosis. Pooled data showing nest building score (scale of 1 to 5) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice Petition 870250075665, dated 08 / 26 / 2025, pp. 199 / 234 36 / 64 treated with NP137 show that the reduced nesting behaviors observed in Endo mice treated with Ab control are normalized with NP137 treatment. Data represent mean ± SEM with NS = not significant and *P < 0.05, Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, from N = 14 to 16 mice per treatment group. Each point represents an individual animal.

[00118] Figure 8: Treatment with NP137 tends to reverse thermal cutaneous hypersensitivity developed in the syngeneic mouse model of endometriosis.

[00119] Pooled data showing the latency of reaction to the heating plate (sec) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137. The data show a trend toward increased sensitivity to noxious thermal stimuli exhibited by Endo mice compared to Sham mice treated with Ab control, which is partially reversed in Endo mice treated with NP137. Data represent mean ± SEM with NS = not significant, ordinary one-way ANOVA with Tukey's posthoc multiple comparison test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, from N = 4 to 8 mice per treatment group. Each point represents an individual animal.

[00120] Figure 9: The NP137-induced improvement in the overall well-being of mice with Endo is confirmed in a new cohort of animals. Pooled data displaying nest-building scores (scale of 1 to 5) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137 show that the reduced nesting behaviors observed in mice Petition 870250075665, dated 08 / 26 / 2025, pages 200 / 234 37 / 64 Endo-treated mice with the Ab control are normalized with the NP137 treatment. Data represent the mean ± SEM with NS = not significant and *P < 0.05, Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances, from N = 20 to 21 mice per treatment group. Each point represents an individual animal.

[00121] Figure 10: Treatment with NP137 tends to reduce the size of endometriosis lesions and the number of lesions developed in the syngeneic mouse model of endometriosis.

[00122] Figure 10A) Pooled data show a slight reduction in mean maximum lesion diameter 10 weeks after Endo induction following chronic treatment with NP137 compared to control Ab.

[00123] Figure 10B) Pooled data show a slight reduction in mean lesion size per animal 10 weeks after Endo induction following chronic treatment with NP137 compared to the Ab control.

[00124] Figure 10C) Pooled data show a slight reduction in the number of lesions developed per animal 10 weeks after Endo induction following chronic treatment with NP137 compared to the Ab control. Data represent mean ± SEM. NS, P > 0.05, non-parametric Mann-Whitney test for 2 groups with unequal variances and unpaired TTest for 2 groups with equal variances of N = 5 mice en = 24 to 33 lesions per treatment group.

[00125] Figure 11: NP137-induced reduction in endometriosis lesion size is confirmed in a new cohort of animals.

[00126] Figure 11A1) Representative images of the reproductive tract 10 weeks after induction surgery for a control Sham mouse treated with Ab (top panel, sutures only in the uterine horns and beside the uterus (arrowheads indicate positioning)), Endo mouse (endometrial fragments attached to the uterine horns and to the uterus) Petition 870250075665, dated 08 / 26 / 2025, pages 201 / 234 38 / 64 side of the uterus (arrowheads indicate positioning) treated with Ab control (middle panel) or NP137 (bottom panel).

[00127] Figure 11A2) Representative images of dissected and cleaned endometriosis lesions collected from the uterus and uterine horns of Endo mice treated with Ab control (4 mice, top panel) and NP137 (4 mice, bottom panel).

[00128] Figure 11B) Pooled data show a significant reduction in mean lesion weight 10 weeks after Endo induction following chronic treatment with NP137 compared to the Ab control.

[00129] Figure 11B1) Grouped data displayed as the average weight of all lesions per animal (g).

[00130] Figure 11B2) Grouped data displayed as the average weight of lesions located in the uterus (g).

[00131] Figure 11B3) Grouped data displayed as the average weight of lesions located in the uterine horns (g).

[00132] Figure 11C) Pooled data show a significant reduction in mean maximum lesion diameter 10 weeks after Endo induction following chronic treatment with NP137 compared to the Ab control. Data represent mean ± SEM. NS, P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001, non-parametric Mann-Whitney test for 2 groups with unequal variances of N = 12 to 16 mice en = 44 to 53 lesions per treatment group.

[00133] Figure 11C1) Grouped data displayed as the mean maximum lesion diameter of all lesions per animal (mm).

[00134] Figure 11C2) Grouped data displayed as the mean maximum lesion diameter of lesions located in the uterus (mm).

[00135] Figure 11C3) Grouped data are displayed as the mean maximum lesion diameter of lesions located in the uterine horns Petition 870250075665, dated 08 / 26 / 2025, pages 202 / 234 39 / 64 (mm).

[00136] Figure 12: The reduction in thermal cutaneous hypersensitivity induced by NP137 is confirmed in a new cohort of animals.

[00137] Figure 12A) Pooled data showing the mechanical paw withdrawal threshold induced by stimulation of the von Frey electronic filament (EvF) of the hind paw (g) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137. The data show that the greater sensitivity to mechanical stimuli exhibited by Endo mice, compared to Sham mice treated with Ab control, is normalized with NP137 treatment.

[00138] Figure 12B) Pooled data showing the reaction latency to the heating plate (sec) in Sham mice treated with Ab control, Endo mice treated with Ab control, and Endo mice treated with NP137. The data show that the increased sensitivity to noxious thermal stimuli exhibited by Endo mice compared to Sham mice treated with Ab control is absent in Endo mice treated with NP137. Data represent mean ± SEM with NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, ordinary one-way ANOVA with Tukey's post-hoc multiple comparison test for groups of equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups of unequal variances, from N = 20 to 22 mice per treatment group. 1. Animals

[00139] The Animal Ethics Committee of the Research Institute The South Australian Health and Medical Research Authority (SAHMRI) approved all experiments involving animals (ethics number SAM342). All animal experiments complied with the standards. Petition 870250075665, dated 08 / 26 / 2025, pages 203 / 234 40 / 64 relevant regulatory requirements and the ARRIVE guidelines. Six-week-old female C57BL / 6J mice were used and acquired from an internal C57BL / 6J breeding program (Jax strain no. 000664; originally purchased from The Jackson Laboratory (MP14 breeding barn; Bar Harbor, ME; USA) within SAHMRI's opportunistic and pathogen-free animal care facilities. All female mice used in this study were virgin (never mated) and housed in the absence of males since weaning. Mice were housed individually after surgery in individually ventilated cages (IVCs) filled with dust-free coarse chip poplar bedding (PURA®; Cat no. - ASPJMAEB-CA, Niederglatt, Switzerland). The animal cages were stored in IVC racks in housing rooms within a temperature- and humidity-controlled environment, maintaining 22 ± 1 °C and a cycle of 12 hours of light / 12 hours of darkness.The mice had free access to LabDiet® JL Rat and Mouse / Auto6F food (Cat n° 5K52, St. Louis, MO; USA) and received autoclaved reverse osmosis purified water. 2. Mouse model of surgically induced endometriosis

[00140] For this study, we used an adapted version of our previously established autologous mouse model of surgically induced endometriosis [9] (all content of this article is incorporated here by reference). For this study, a total of 4 uterine horn fragments from donors were surgically sutured along the female reproductive tract, with 2 along the uterus (1 on each side) and 2 attached to the uterine horns (1 in each horn). See Figure 1 for the chemical diagram showing the timeline of the different interventions performed in this study. 2.1 Ovariectomy Surgery

[00141] Seven days before surgical induction of endometriosis, female mice were ovariectomized to deplete the Petition 870250075665, dated 08 / 26 / 2025, pages 204 / 234 41 / 64 endogenous steroid production. Briefly, mice were anesthetized with isoflurane (2.5% / 0.5 LO 2) and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before the start of surgery. After aseptic conditions, a small longitudinal paralumbar incision of the skin and dorsal abdominal muscle was made to expose the ovaries on both sides. The ovaries were exteriorized and the oviduct / ovarian artery and ovarian vein / ligaments were sectioned by cauterization. The uterine horns were then returned to the abdominal cavity and the superficial layer of skin was closed using 9 mm clips (AutoClip® System, FST). The ovariectomized mice were allowed to recover for a minimum of 5 days before removal of the surgical clips. Since endometriosis is an estrogen-dependent disease, it is important to maintain stable circulating estrogen levels and minimize any differences related to the stage of the estrous cycle.Considering these factors, all mice received an intraperitoneal injection of 100 μg / kg of estradiol benzoate (Progynon-B) after ovariectomy, which continued weekly throughout the study. 2.2 Endometriosis (Endo) and Sham Surgeries

[00142] Seven days after ovariectomy, experimental mice were surgically induced with endometriosis using uterine horn tissue collected from donor mice. To collect the donor tissue, the uterine horns were excised and fixed in a dissection plate with chilled sterile phosphate-buffered saline (PBS) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) (Sigma-Aldrich, St. Louis, MO). The uterine horns were opened longitudinally, with 4 x 2 mm sections of uterine horn tissue collected using a 2 mm biopsy punch (Kai medical, KAI00010). The experimental recipient mice were then anesthetized with isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before the start of surgery. Following Petition 870250075665, dated 08 / 26 / 2025, pages 205 / 234 Under aseptic conditions (42 / 64), a small midline abdominal incision was made to expose the abdominal cavity, and 2 mm circular fragments of donor uterine horn tissue were sutured along the uterus (2 fragments) and uterine horns (1 fragment in each horn) using Prolene 6.0 sutures. For sham surgeries, a similar length of Prolene 6.0 sutures was attached to the same surgical sites as in the endo surgery, but in the absence of any tissue. The abdominal muscle was then sutured and closed, and the superficial skin layer closed using 9 mm clips (AutoClip®System, FST). Throughout the surgery and during the recovery period, the animals were positioned on a heating mat to maintain body temperature and monitored daily for complications. No signs of distress or unusual pain behavior were observed during recovery. 2.3 Mouse model of syngeneic endometriosis

[00143] In this study, we used a second endometriosis model, called the syngeneic endometriosis mouse model. This clinically relevant mouse model is generated by inoculating uterine horn fragments into the peritoneal cavity, as described in the publication incorporated here by reference

[21] . This is a minimally invasive model that resembles the natural occurrence of endometriosis lesions in humans, due to the fact that the inoculated fragments are randomly distributed throughout the peritoneal cavity, where they implant and develop into lesions

[21] . This model developed lesions and generalized chronic pelvic pain, characteristic of endometriosis in humans. 2.4 Endometriosis (Endo) and Sham Surgery for the Syngeneic Model

[00144] Seven days after ovariectomy, experimental mice were induced with endometriosis by inoculation of uterine horn tissue fragments collected from donor mice into a Petition 870250075665, dated 08 / 26 / 2025, pp. 206 / 234 43 / 64 recipient mice. To collect donor tissue, the uterine horns were excised and fixed in a dissection plate with 0.5 ml of ice-cold sterile phosphate-buffered saline (PBS) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) (Sigma-Aldrich, St. Louis, MO). The uterine horns were opened longitudinally and cut into small fragments. The experimental recipient mice were then anesthetized with isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before the start of induction. Following aseptic conditions, a small midline abdominal incision was made to expose the abdominal cavity, and half of the collected uterine horn tissue fragments from the donor were inoculated, using a 1 ml pipette, into the peritoneal cavity of a recipient mouse (total volume of ~0.2 ml of solution containing fragments).For sham surgeries, a similar procedure was performed, but 0.2 ml of sterile phosphate-buffered saline (PBS) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) (Sigma-Aldrich, St. Louis, MO) was inoculated. The abdominal muscle was then sutured and closed, and the superficial skin layer was closed using 9 mm clips (AutoClip® System, FST). During surgery and the recovery period, the animals were placed on a thermal blanket to maintain body temperature and monitored daily for complications. No signs of distress or unusual pain behaviors were observed during recovery. 3. Characterization of the growth and development of endometriosis lesions.

[00145] Endometriosis lesions that grew from surgically transplanted uterine horn fragments were collected and measured at the end of all behavioral trials to monitor the development of endometriosis lesions after antibody treatment. Petition 870250075665, dated 08 / 26 / 2025, pages 207 / 234 44 / 64 control or NP137. All tissue was collected at least 24 hours after the last antibody control or NP137 treatment to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen variation between time points. 3.1 Collection and measurement of endometriotic lesions

[00146] Lesions were located, dissected, measured, and weighed post-mortem in Endo (control Ab) and Endo (NP137) mice. A total of 4 endometriosis lesions were collected from each mouse (2 uterine lesions and 2 uterine horn lesions). Lesions damaged during dissection or that could not be separated from the surrounding tissue were not included in the lesion growth analysis. Measurements at the widest point of the lesion were taken with calipers (8.5 cm / 3-1 / 4” Castroviejo calipers). 3.2. Statistical analysis of lesion growth

[00147] Data are presented as mean ± SEM, N represents the number of animals and n represents the number of individual lesions per group. Data were graphically represented using Prism 9 software (GraphPad Software, San Diego, CA, USA). Data were statistically analyzed using the non-parametric Mann-Whitney test for two groups with unequal variances. Differences were considered statistically significant at ** P < 0.01, *** P < 0.001, **** P < 0.0001. 4. In vivo assessment of chronic pelvic pain by quantifying visceromotor responses (VMR).

[00148] The visceromotor response (VMR) is a nociceptive reflex of the brainstem that consists of the contraction of abdominal muscles in response to noxious distension of hollow organs, such as the vagina and colorectal area [4, 6, 7, 19, 25, 27, 16]. We recorded the VMR for vaginal distension. Petition 870250075665, dated 08 / 26 / 2025, pages 208 / 234 45 / 64 (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, we previously showed that mice with endometriosis develop colonic and vaginal hypersensitivity [9, 21]. To determine whether chronic treatment with NP137 was able to reduce the vaginal and colonic hypersensitivity developed in a mouse model of endometriosis, we used VMR to determine any differences between Sham (Ab control) and Endo (Ab or NP137 control) mice. All mice were studied at least 24 hours after the last Ab or NP137 control treatment to minimize any acute effects of either treatment and at least 3 days after estradiol administration to minimize estrogen variation between time points. 41. Surgical implantation of electromyography (EMG) electrodes

[00149] VMR is objectively assessed by recording the electrical activity (electromyography (EMG)) produced by abdominal muscle contractions in response to non-noxious and noxious vaginal or colorectal distensions. To enable this, three days prior to VMR and under isoflurane anesthesia, the bare ends of two Teflon-coated stainless steel wires (Advent Research Materials Ltd, Oxford, UK) were sutured to the right external oblique muscle of the abdomen of Sham or Endo mice and tunneled subcutaneously to be exteriorized at the base of the neck for future access. At the end of the surgery, the mice received prophylactic antibiotic (Baytril®; 5 mg / kg sc) and analgesic (buprenorphine; 0.5 mg / 10 kg sc) and returned to their individual housings and were left to recover for at least three days, as previously described [21, 9, 8]. 4.2 Assessment of visceromotor responses (VMR) to vaginal distension (VD) or colorectal distension (CRD)

[00150] On the day of the VMR assessment, the mice were Petition 870250075665, dated 08 / 26 / 2025, pp. 209 / 234 46 / 64 were temporarily anesthetized with inhaled isoflurane and a saline enema administered via catheter into the colon and rectum (100 μL) or vaginal cavity (50 μL), and a balloon was inserted into the colon and rectum or vaginal cavity for VMR assessment, as previously described [21, 9, 6, 7]. Briefly, a lubricated balloon was gently introduced through the anus and inserted into the colon and rectum to 0.25 cm beyond the anal verge (2.5 cm long latex balloon) for CRD or gently passed through the vagina and inserted to 1 mm proximal to the vaginal verge (3 mm long latex balloon) for VD. 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, pressure-controlled balloon distension. The mice were gently restrained in a restraint device with dorsal access and allowed to recover from anesthesia for 10 minutes before the distension sequence began.The distension sequences were predefined and applied via barostat at 20-40-50-60-70-80 mm Hg (20-second duration) at 4-minute intervals for CRD or 20-30-40-60-70-80 mm Hg (30-second duration) applied at 3-minute intervals for VD. The EMG electrodes were relayed to a data acquisition system and the signal was recorded (NL100AK stage), amplified (NL104), filtered (NL 125 / 126, Neurolog, Digitimer Ltd, 50-5000 Hz passband) and digitized (CED 1401, Cambridge Electronic Design, Cambridge, UK) to a PC for offline analysis using Spike2 (Cambridge Electronic Design), as previously described [6]. The mice were allowed to recover from their first distension protocol (randomized to VD or CRD) for a minimum of 3 hours before the next distension protocol. After this final distension, the mice were returned to their IVC cages and support for the final ip treatment and tissue collection. 4.3 Statistical analysis of VMR for VD and CRD

[00151] The analog EMG signal was rectified, integrated, and used for VMR analysis. To quantify the magnitude of VMR in each Petition 870250075665, dated 08 / 26 / 2025, pages 210 / 234 47 / 64 distension pressure, the area under the curve (AUC) during distension was corrected for baseline activity (pre-distension AUC). The AUC was quantified by calculating the AUC of the EMG signal at each distension pressure. Additionally, the sum of all AUCs for all combined distension pressures (total AUC) is also analyzed as an overall parameter of VMR responses for each individual mouse. VMR data are presented as mean ± SEM, and N represents the number of animals. The analysis and figures were prepared using GraphPad Prism software (version 9, San Diego, CA, USA). VMR data for AUC at each distension pressure were statistically analyzed using generalized estimating equations (GEE), followed by the least significant difference (LSD) post-hoc test, when appropriate, using SPSS 23.0.Data for total AUC were statistically analyzed using Prism 9 software (GraphPad Software, San Diego, CA, USA) and, where appropriate, using ordinary one-way ANOVA with Sidak's post-hoc multiple comparison test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 5. In vivo assessment of cutaneous sensitivity evoked by thermal and mechanical stimuli.

[00152] To determine whether chronic treatment with NP137 was able to reduce evoked pain-like behaviors developed in a mouse model of endometriosis, we used both the hot plate test (thermal sensitivity) and the von Frey electronic test (EvF) (mechanical sensitivity) to assess cutaneous sensitivity. To ensure consistency and reduce bias, the same researcher performed all measurements blindly. All mice were studied for at least Petition 870250075665, dated 08 / 26 / 2025, pp. 211 / 234 48 / 64 hours after the last control treatment with antibody or NP137 to minimize any acute effects from either treatment, and at least 3 days after estradiol administration to minimize estrogen variation between time points. 5.1 Assessment of cutaneous sensitivity to mechanical stimuli using the VON FREY ELECTRONIC REFLEX (VEF)

[00153] The von Frey electronic test (EvF) is used to assess mechanical allodynia to mechanical stimuli by measuring withdrawal to a stimulus that is not normally painful

[12] . We 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 habituated to the enclosure made of transparent Plexiglas (observation chamber approx. 230x240x146 mm, BSBIOPVF, Panlab, Spain) and placed on an elevated wire mesh support for 30 minutes each day for 2 days. On the day of the test, the mice were moved to the test room in their IVC and allowed to acclimate for a minimum of 15 minutes. The animals were placed individually in the test arenas, with an aerated lid placed on top, and left to rest for 15 minutes. The EvF unit (portable force transducer, BSBIOEVF4s, Panlab, Spain) was equipped with a semi-flexible, zeroed tip.Once the animal was still and quiet, the force transducer was applied perpendicularly to the animal's hind paw or lower abdomen below. The force was gradually increased until a clear withdrawal response was observed. The maximum force applied (in grams) that caused the paw or abdomen to withdraw was recorded as the withdrawal threshold. Each area was stimulated 5 times, with the average recorded as the experimental withdrawal threshold for each mouse. At the end of the test, the mice were returned to their cage and to the IVC stand. Petition 870250075665, dated 08 / 26 / 2025, pp. 212 / 234 49 / 64 Statistical analysis

[00154] 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 were analyzed, where appropriate, using an ordinary one-way ANOVA with Tukey's post-hoc multiple comparison test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 5.2 Assessment of skin sensitivity to noxious thermal stimuli with hot plate latency

[00155] The hot plate test is a popular test used to measure acute thermal nociception by monitoring reflexive behaviors to thermal stimuli. Using this technique, we previously demonstrated the development of thermal hypersensitivity in a mouse model of endometriosis [21,9]. Prior to the test, mice were moved to the test room in their IVC and allowed to acclimate for a minimum of 15 minutes. Mice were then individually placed on a pre-heated hot plate (LE7406, Harvard Apparatus, Panlab, Spain), set to 54 ± 1 °C, which is shown to be a noxious stimulus

[12] , and surrounded by a transparent methacrylate protective cover. The time between being placed on the hot plate and their first sign of pain response, including licking the hind paw, snapping the hind paw, or jumping (whichever occurred first), was recorded as hot plate latency.The mice were allowed to recover in their cage for 24 hours, and the test was repeated. The average response latency of both tests was recorded as the final latency of the hot plate. Petition 870250075665, dated 08 / 26 / 2025, pages 213 / 234 50 out of 64 mice were then returned to the cage and the IVC stand. Statistical analysis

[00156] The mean response, in seconds, obtained in both exposures to the heating plate 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 were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 6. In vivo assessment of spontaneous behavior

[00157] To determine whether chronic treatment with NP137 was able to reduce the increase in spontaneous pain behaviors, similar to those developed in a mouse model of endometriosis, we used voiding pattern analysis along with nighttime nest building analysis to determine any differences between Sham (Ab control) and Endo (Ab or NP137 control) mice. All mice were evaluated at least 24 hours after the last Ab or NP137 control treatment to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen variation between time points. To ensure consistency and reduce bias, the same researcher performed all measurements blindly. At the end of each test protocol, the mice were returned to their cage and IVC support for subsequent assays. 6.1 In vivo assessment of bladder dysfunction by analysis of the emptying pattern

[00158] The analysis of the emptying pattern is a Petition 870250075665, dated 08 / 26 / 2025, pages 214 / 234 51 / 64 voiding assessment tool that provides information on changes in spontaneous behavioral patterns related to 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 assess changes in bladder function after the development of endometriosis in mice [9, 21].

[00159] Voiding pattern analysis was performed on all mice 8 weeks after endometriosis induction surgery or Sham and 4 weeks after treatment with Ab or NP137 control. For this, all bedding was removed from the cage of each Endo and Sham mouse and filter paper was placed at the bottom of each cage. The mice remained in their lined cages for 3 hours, between 9 am and 12 pm, with free access to food and water. During this time, the mice produced spontaneous voiding patterns on the filter paper. The filter paper was collected and stored for imaging, the bedding was returned to the IVCs and the mice returned to the IVC stand.

[00160] Urine collected on filter paper was imaged using an ultraviolet 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 predefined limits in ImageJ software and, for the purposes of this study, the number of small (100-1,000 pixels), medium (1,001-100,000 pixels) and large (>100,000 pixels) spots were quantified. Statistical analysis

[00161] Data are presented as mean ± SEM, and N represents the number of animals. Data were graphically represented using Prism 9 software (GraphPad Software, San Diego, CA, USA) and, where appropriate, analyzed using an ordinary one-way ANOVA with Petition 870250075665, dated 08 / 26 / 2025, pages 215 / 234 52 / 64 Tukey's post-hoc multiple comparison test for groups with equal variances; or Kruskal-Wallis test with Dunn's multiple comparisons for groups with unequal variances. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 6.2. Assessment of overall animal welfare through SPONTANEOUS ANALYSIS OF NOCTURNAL NESTING

[00162] Spontaneous nesting analysis is a sensitive assessment tool that provides information on changes in spontaneous behavioral patterns related to overall well-being in mice

[13] . In this study, we performed a nocturnal nesting analysis using a predefined 5-point scale to assess the effect of chronic NP137 dosing on the overall well-being of the animals, as previously described

[11] .

[00163] Nesting pattern analysis was performed on all mice 8 weeks after endometriosis or sham induction surgery and 4 weeks of treatment with Ab control or NP137. For this, all environmental enrichment items (including the original nest material, cardboard tunnels, and fabric) were removed from each Endo and Sham mouse cage, leaving only the original bedding material at the base of each cage. A pre-weighed Nestlet (approximately 3 g 5 cm squares of cotton) was placed in each cage, and the mice were returned to the IVC stand and left undisturbed to nest overnight (from 4 pm to 9 am).The following morning, the individual nests were blindly assessed and rated on a predetermined 5-point nest rating scale, ranging from 1 to 5, with a score of 1 reflecting an untouched nest and a score of 5 representing a fully torn nest with high, fluffy walls, as previously described. Petition 870250075665, dated 08 / 26 / 2025, pages 216 / 234 53 / 64

[11] . The environmental enrichment was returned to the cage and the mice were returned to their IVC supports. Statistical analysis

[00164] Data are presented as mean ± SEM, and N represents the number of animals. Data were graphically represented using Prism 9 software (GraphPad Software, San Diego, CA, USA) and analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons. Differences were considered statistically significant at * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 7. Results and conclusions 7.1. Mice with endometriosis developed cyst-like endometriotic lesions.

[00165] The mouse model of endometriosis used in this study is an adaptation of the previously established model of endometriosis induced by surgical transplantation [9]. In this model, we transplanted fragments of the uterine horn on each side of the uterine dome and at the tip of the uterine horns, as detailed in the methods section (also Figure 2A1). Control mice (sham mice) were generated following the same surgical procedure, but in the absence of uterine horn tissue (Figure 2A1). As described previously [9], the transplanted fragments developed endometriotic lesions 10 weeks after transplantation (also Figure 2A1). 7.2. Treatment with NP137 reduced the development of endometriotic lesions.

[00166] To investigate whether treatment with NP137 could limit lesion development and provide analgesia to mice with endometriosis, we examined the growth of endometrial lesions. The efficacy of NP137 treatment was quantified by comparing the degree of development Petition 870250075665, dated 08 / 26 / 2025, pp. 217 / 234 54 / 64 of endometriosis in Endo mice treated with NP137 compared to that observed in Endo mice treated with the control antibody NP001 (Ab control) (Figure 2A1). As previously described, Sham mice do not develop endometrial lesions at the surgical suture sites (Figure 2A1).

[00167] Examination of the female reproductive tract of mice with endometriosis shows that chronic treatment with NP137 reduced the size of the lesions developed (Figure 2A1-2). The reduction in lesion size is visually evident in Figure 2A2, which shows endometrial lesions collected from 4 Endo mice treated with NP001 (control Ab) (Figure 2A2, left panel) and endometrial lesions collected from 3 Endo mice treated with NP137 (Figure 2A2, right panel). Quantitative analysis of the collected lesions shows that treatment with NP137 significantly reduced the weight and size of these endometriotic lesions (Figure 2B-C). Interestingly, the NP137-induced reduction in lesion size was specific to lesions developed around the uterus, and not to those growing at the ends of the uterine horns (Figure 2A2 and 2B-C).Notably, a separate set of treatments (4 additional Endo mice treated with NP001 and 5 Endo mice treated with NP137) confirmed the initial findings showing the ability of NP137 to significantly reduce lesion size in mice with Endo (Figure 11A1-2). Furthermore, the inclusion of data generated by these additional mice in the overall data (Figure 11BC) eliminates the regional differences observed in the first set of treatments (Figure 2A2 and 2B-C). Therefore, NP137 is effective in reducing the development of endometriosis lesions, regardless of where they developed (Figure 11B2-3 and 11C2-3).

[00168] Interestingly, we also tested the effect of NP137 on reducing lesions developed in a different mouse model. Petition 870250075665, dated 08 / 26 / 2025, pages 218 / 234 55 / 64 of endometriosis known as the syngeneic mouse model of endometriosis (Figure 10). This is a minimally invasive model that resembles the natural occurrence of endometriosis lesions in humans due to the fact that the inoculated fragments are randomly distributed throughout the peritoneal cavity, where they implant and develop into lesions. Notably, treatment with NP137 tends to reduce the size and number of lesions developed within the peritoneal cavity of the syngeneic mouse model of endometriosis (Figure 10A-C). This suggests that NP137 is not only efficient in reducing the growth of lesions once formed, but potentially prevents the formation of the lesions themselves. 7.3. Treatment with NP137 reduced the vaginal hyperalgesia that developed. In endometriosis.

[00169] Visceral pain was measured in vivo by recording the visceromotor pain response (VMR) evoked by vaginal distension (VD). VMR is the gold standard for measuring pain from hollow organs, such as the vagina [3, 26] and colon (as per our previous studies [6, 16]). VMR responses were quantified by recording electromyographic (EMG) activity from electrodes surgically implanted in the abdominal muscles of mice, as previously described [8].

[00170] In Sham mice, vaginal distension increases VMR responses, with the degree of VMR related to the applied pressure (Figure 3A). We found that mice with fully developed endometriosis showed a significant increase in VMR responses to VD at 20 and 40-70 mmHg (Figure 3A) and to all distensions combined (Figure 3B); indicating the development of vaginal hyperalgesia in mice with endometriosis. Notably, compared to Endo mice treated with Ab control, treatment with NP137 significantly reduced the VMR response in Endo mice, specifically Petition 870250075665, dated 08 / 26 / 2025, pages 219 / 234 56 / 64 at lower distending pressures (<40 mmHg) (Figure 3C). Furthermore, the overall effect of NP137 was to normalize VMR responses to those exhibited by Sham control mice (Figure 3D).

[00171] Overall, these results demonstrate that treatment with the monoclonal antibody NP137 is an effective approach to alleviate vaginal hyperalgesia associated with endometriosis. 7.4. Treatment with NP137 reduced colorectal hyperalgesia developed in endometriosis.

[00172] Women with endometriosis are commonly co-diagnosed with other chronic visceral comorbidities, such as irritable bowel syndrome (IBS)

[29] . Next, we aimed to determine whether mice with fully developed endometriosis also exhibit increased sensitivity to pain induced by colorectal distension (CRD), and whether treatment with NP137 was able to provide analgesia.

[00173] We found that, compared to Sham mice treated with Ab control, Endo mice treated with Ab control exhibited significant hypersensitivity to CRD at all tested distensions of 20-80 mmHg (Figure 4A) and at all distensions combined (Figure 4B); indicating the development of colorectal hyperalgesia in mice with endometriosis. Similar to what was observed with VD when compared to Endo mice treated with Ab control, treatment with NP137 significantly reduced the VMR response in Endo mice, specifically at lower distension pressures (<40 mmHg) (Figure 4C). Furthermore, the overall effect of NP137 was to normalize VMR responses to those exhibited by Sham control mice (Figure 4D).

[00174] It is important to emphasize that these findings are clinically relevant, as women with endometriosis frequently Petition 870250075665, dated 08 / 26 / 2025, pages 220 / 234 57 / 64 present with severe CPP involving the colon and vagina [20, 29]. Overall, these results show that treatment with the anti-netrin monoclonal antibody, NP137, is an effective approach to alleviate colorectal hyperalgesia associated with endometriosis. 7.5. Treatment with NP137 normalized the altered bladder function developed in endometriosis.

[00175] Women with endometriosis frequently suffer from visceral comorbidities, including overactive bladder (OAB) and interstitial cystitis / painful bladder syndrome (CI-PBS). In this study, we aimed to determine whether our mouse model of endometriosis developed altered bladder function and, primarily, whether treatment with NP137 could normalize function. To investigate this, we examined and compared the spontaneous bladder emptying patterns of Sham and Endo mice treated with Ab control with Endo mice treated with NP137, as an indication of altered bladder function.

[00176] We found that, compared to Sham mice, mice with fully developed endometriosis showed alterations in bladder emptying patterns, evidenced by an increase in the total number of urine spots (Figure 5A). This interrupted and scattered emptying pattern, characterized by an increase in the number of small urine spots (Figure 5B), is indicative of the urgency and frequency symptoms of OAB / IC-PBS

[17] .

[00177] Interestingly, treatment with NP137 normalized bladder dysfunction developed in mice with endometriosis, as evidenced by a reduction in the total number of urine stains (Figure 5A) observed in Endo mice treated with NP137, specifically the small urine stains (Figure 5B). Furthermore, the overall effect of NP137 was to normalize voiding patterns for those Petition 870250075665, dated 08 / 26 / 2025, pages 221 / 234 58 / 64 displayed by the Sham control mice (Figure 5A-B). 7.6. Treatment with NP137 normalizes the increased skin sensitivity to thermal and mechanical stimuli developed in endometriosis.

[00178] In addition to suffering from comorbidities associated with visceral organs, women with endometriosis may also present with altered cutaneous sensitivity [18, 28]. Here, we determined whether our mouse model of endometriosis exhibited altered cutaneous sensitivity and whether treatment with NP137 could correct it. To determine whether the development of endometriosis influences cutaneous sensitivity evoked by thermal and mechanical stimuli, mice were subjected to hot plate and electronic von Frey (EvF) tests.

[00179] We found that mice with fully developed endometriosis treated with Ab control exhibited increased sensitivity to mechanical stimulation of their hind paw, indicated by a significantly reduced force required to elicit a response compared to their Sham counterparts (Figure 6A). The force required to elicit a response is termed the mechanical threshold, with a reduction in this threshold indicating increased sensitivity to mechanical stimuli. We also found that Ab control Endo-treated mice exhibited increased sensitivity to thermal stimuli, indicated by a significantly reduced response latency to the hot plate compared to their Sham counterparts (Figure 6B). These results indicate that our mouse model of endometriosis does indeed experience increased sensitivity to cutaneous mechanical and thermal stimuli compared to Sham mice.

[00180] Interestingly, we discovered that treatment with NP137 efficiently reversed the increased sensitivity to mechanical and thermal stimuli observed in Endo mice treated with Petition 870250075665, dated 08 / 26 / 2025, pages 222 / 234 59 / 64 antibody control (Figure 6A-B). Importantly, the observed reduction was to levels similar to those observed in Sham mice treated with antibody control (Figure 6A-B). Notably, a separate set of treatments (6 more Endo mice treated with NP001 and 6 Endo mice treated with NP137) confirmed the initial findings, showing the ability of NP137 to significantly reduce mechanical and thermal cutaneous hypersensitivity in mice with Endo (Figure 12A-B).

[00181] Furthermore, we also found that mice with Endo-induced by inoculation of uterine horn fragments into the peritoneal cavity (the syngeneic mouse model) exhibited a slightly increased sensitivity to thermal stimuli, indicated by a slight reduction in the latency of the hot plate response, compared to their sham counterparts (Figure 8). Interestingly, we found that treatment with NP137 tends to reverse this effect observed in Endo mice treated with control antibody (Figure 8). 7.7. NP137 IMPROVES SIGNS OF REDUCED GENERAL WELL-BEING PRESENTED IN ENDOMETRIOSIS

[00182] Finally, we tested whether the development of endometriosis alters spontaneous behavior in a way that indicates a deterioration in the animal's overall well-being and whether treatment with NP137 could help maintain the animal's overall well-being. Analysis of spontaneous nest building is a sensitive assessment tool that provides information on changes in spontaneous behavioral patterns related to overall well-being in mice

[13] .

[00183] In this study, we analyzed the nocturnal nesting behavior of Sham and Endo mice treated with Ab control and found that mice with fully developed endometriosis had a reduced ability to build their nest during this period. Petition 870250075665, dated 08 / 26 / 2025, pages 223 / 234 60 / 64 (Figure 7). This reduction in nest building indicates a lack of nocturnal activity, suggesting that the animal's well-being is reduced by the development of endometriosis. We found that treatment with NP137 partially recovers nesting behaviors in mice with endometriosis (Figure 7), with Endo mice treated with NP137 exhibiting nesting behavior similar to that shown by Sham mice (Figure 7). Notably, a separate set of treatments (6 additional Endo mice treated with NP001 and 5 Endo mice treated with NP137) confirmed the initial findings showing the ability of NP137 to significantly improve the overall well-being of mice with Endo (Figure 9). The inclusion of the data generated by these additional mice in the overall data showed that treatment with NP137 completely reversed the nocturnal nesting deficiency generated in mice with Endo.This is evident from the fact that mice with Endo treated with NP137 exhibited nesting behavior patterns similar to those exhibited by sham mice treated with NP001 (Figure 9), indicating its beneficial effect in reducing anxious behaviors associated with endometriosis. 7.8. Conclusion

[00184] Overall, the results of this preclinical study demonstrate that chronic treatment with NP137, a clinically approved anti-netrin-1 monoclonal antibody, is effective in reducing the size of endometriotic lesions developed in a clinically relevant mouse model of endometriosis. Furthermore, treatment with NP137 was able to reverse altered pain sensitivity in multiple peripheral organs, reducing chronic pelvic pain experienced by mice with fully developed endometriosis. These results strongly suggest that NP137 is a viable therapeutic strategy to provide reduction or Petition 870250075665, dated 08 / 26 / 2025, pp. 224 / 234 61 / 64 disappearance of endometriosis lesions and relief of chronic pelvic pain in women with endometriosis. References 1. Adaes, S. et al., Glycal activation in the collagenase model of nociception associated with osteoarthritis. Mol Pain, 2017. 13: p. 1744806916688219. 2. Berkley, K.J., A. Cason, H. Jacobs, H. Bradshaw, and E. Wood, Vaginal hyperalgesia in a rat model of endometriosis. Neurosci Lett, 2001. 306(3): p. 185-8. 3. Berkley, KJ et al., Endometrial-induced vaginal hyperalgesia in rats: effect of stropause, ovariectomy, and estradiol replacement. Pain, 2007. 132 Suppl 1: p. S150-9. 4. Carstens, BB et al., Structure-Activity Studies of Cysteine- Rich alpha-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABA(B) Receptor Activation Reveal a Minimal Functional Motif. Angew Chem Int Ed Engl, 2016. 55(15): p. 4692-6. 5. Cason, A.M., C.L. Samuelsen, and K.J. Berkley, Estrous changes in vaginal nociception in a rat model of endometriosis. Horm Behav, 2003. 44(2): p. 123-31. 6. Castro, J. et al., Cyclic analogues of alpha-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain. Br J Pharmacol, 2018. 175(12): p. 2384-2398. 7. Castro, J. et al., Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity. JCI Insight, 2019. 4(20). 8. Castro, J. et al., Pharmacological modulation of voltage- gated sodium (NaV) channels alters nociception arising from the female reproductive tract. Pain, 2020. Petição 870250075665, de 26 / 08 / 2025, pág. 225 / 234 62 / 64 9. Castro, J. et al., A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities. FASEB J, 2021.35(4): p. e21430. 10. Cozzolino, M. et al., Variables Associated with Endometriosis-related Pain: A Pilot Study using a Visual Analogue Scale. Rev Bras Ginecol Obstet, 2019. 41(3): p. 170-175. 11. Deacon, R.M., Assessing nest building in mice. Nat Protoc, 2006. 1(3): p. 1117-9. 12. Deuis, J.R., L.S. Dvorakova, and I. Vetter, Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci, 2017. 10: p. 284. 13. Gaskill, B.N., A.Z. Karas, J.P. Garner, and K.R. PritchettCorning, Nest building as an indicator of health and welfare in laboratory mice. J Vis Exp, 2013(82): p. 51012. 14. Ge, P. et al., Linaclotide treatment reduces endometriosisassociated vaginal hyperalgesia and mechanical allodynia through viscerovisceral cross-talk. Pain, 2019. 15. Grandin, M. et al., Structural Decoding of the Netrin-1 / UNC5 Interaction and its Therapeutical Implications in Cancers. Cancer Cell, 2016. 29(2): p. 173-85. 16. Grundy, L. et al., Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction. JCI Insight, 2018. 3(19). 17. Hill, W.G. et al., Void spot assay: recommendations on the use of a simple micturition assay for mice. American Journal of Physiology-Renal Physiology, 2018. 315(5): p. F1422-F1429. 18. Jarrell, J., L. Malekzadeh, H. Yang, and L. Arendt-Nielsen, The Significance of Cutaneous Allodynia in a Woman With Chronic Pelvic Pain. J Obstet Gynaecol Can, 2015. 37(7): p. 628-632. Petição 870250075665, de 26 / 08 / 2025, pág. 226 / 234 63 / 64 19. Jimenez-Vargas, N.N. et al., Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. Proc Natl Acad Sci U S A, 2018. 115(31): p. E7438-E7447. 20. Maddern, J., L. Grundy, J. Castro, and S.M. Brierley, Pain in Endometriosis. Front Cell Neurosci, 2020. 14: p. 590823. 21. Maddern, J. et al., A syngeneic inoculation mouse model of endometriosis that develops multiple comorbid visceral and cutaneous pain like behaviours. Pain, 2022. 163(8): p. 1622-1635. 22. McAllister, S.L. et al., Endometriosis-induced vaginal hyperalgesia in the rat: role of the ectopic growths and their innervation. Pain, 2009. 147(1-3): p. 255-64. 23. Mehlen, P. and C. Furne, Netrin-1: when a neuronal guidance cue turns out to be a regulator of tumorigenesis. Cell Mol Life Sci, 2005. 62(22): p. 2599-616. 24. Nagabukuro, H. et al., Influence of endometriosis on visceromotor and cardiovascular responses induced by vaginal distention in the rat. Pain, 2007. 132 Suppl 1: p. S96-103. 25. Ness, T.J. et al., Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat. Brain Res, 1988. 450(1-2): p. 153-69. 26. Pierce, A.N. et al., Neonatal vaginal irritation results in longterm visceral and somatic hypersensitivity and increased hypothalamic-pituitaryadrenal axis output in female mice. Pain, 2015. 156(10): p. 2021-31. 27. Salvatierra, J. et al., NaV1.1 inhibition can reduce visceral hypersensitivity. JCI Insight, 2018. 3(11). 28. Slater, H. et al., Heightened cold pain and pressure pain sensitivity in young female adults with moderate-to-severe menstrual pain. Pain, 2015. 156(12): p. 2468-78. Petição 870250075665, de 26 / 08 / 2025, pág. 227 / 234 64 / 64 29. 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Claims

1 / 3 Claims 1. USE OF AN ANTI-NETRIN-1 ANTIBODY OR AN ANTIGEN-BINDING FRAGMENT THEREOF, characterized in that it is used in the manufacture of a medicament for the treatment of endometriosis, wherein the treatment comprises a reduction of endometriosis lesions, especially uterine and / or uterine horn endometriosis lesions.

2. USE, according to claim 1, characterized in that said treatment also includes relief of chronic pelvic pain.

3. USE, according to any of claims 1 to 2, characterized in that said treatment further includes relief of vaginal hyperalgesia.

4. USE, according to any of claims 1 to 3, characterized by the treatment further comprising: - a reduction in colorectal hyperalgesia developed in endometriosis; - an improvement in bladder dysfunction developed in endometriosis; - a reduction in increased skin sensitivity to thermal and / or mechanical stimuli developed in endometriosis; - an improvement in general well-being altered by endometriosis.

5. USE, according to any one of claims 1 to 4, characterized by the antibody or antigen-binding fragment specifically binding to a polypeptide of the sequence SEQ ID NO:

33.

6. USE, according to any of claims 1 to 5, characterized in that the antibody is a monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or fragment comprises a variable VH domain comprising: Petition 870250075665, dated 08 / 26 / 2025, p.229 / 234 2 / 3 - an H-CDR1 with a sequence defined as SEQ ID NO: 5; - an H-CDR2 with a sequence defined as SEQ ID NO: 6; - an H-CDR3 with a sequence defined as SEQ ID NO: 7; a variable domain VL comprising: - an L-CDR1 with a sequence defined as SEQ ID NO: 8; - an L-CDR2 with a sequence YAS; - an L-CDR3 with a sequence defined as SEQ ID NO: 9; or a variable domain VH comprising: - an H-CDR1 with a sequence defined as SEQ ID NO: 28; - an H-CDR2 with a sequence defined as SEQ ID NO: 29; - an H-CDR3 with a sequence defined as SEQ ID NO: 30; a variable domain VL comprising: - an L-CDR1 with a sequence defined as SEQ ID NO: 31; - an L-CDR2 with a sequence defined as SEQ ID NO: 32; - an L-CDR3 with a sequence defined as SEQ ID NO:

9.

7. USE, in accordance with any of the claims in Petition 870250075665, dated 08 / 26 / 2025, p. 230 / 234 3 / 3 6, characterized in that 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 NO: 27 and 19, SEQ ID NO: 20 and 14, SEQ ID NO: 21 and 15, SEQ ID NO: 22 and 16, SEQ ID NO: 23 and 17, SEQ ID NO: 24 and 17, SEQ ID NO: 25 and 16, SEQ ID NO: 26 and 17, SEQ ID NO: 22 and 17, SEQ ID NO: 25 and 18, SEQ ID NO: 21 and 16, and preferably the antibody or fragment thereof comprises a pair of VH and VL sequences from SEQ ID NO: 22 and 16.

8. USE, according to any one of claims 1 to 7, characterized by comprising a human IgG1 constant heavy chain (CH) and / or a human IgG1 constant light chain (CL), in particular a human kappa constant domain.

9. USE, according to any one of claims 1 to 8, characterized by comprising a VH of the sequence SEQ ID NO: 22 and a VL of the sequence SEQ ID NO:

16.

10. USE, according to claim 9, further characterized by comprising a modified human IgG1 CH Genbank AEL33691.1 R97K and a human Kappa IgG1 CL Genbank CAC20459. Petition 870250075665, dated 08 / 26 / 2025, pp. 231 / 234

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