Anti-axon director-1 monoclonal antibodies for treatment of endometriosis and associated pain

Abstract

The present invention relates to the treatment of endometriosis using an anti-axon guiding factor-1 antibody, or an antigen-binding fragment thereof, and the antibody, or a fragment thereof, for use in the treatment of endometriosis. The treatment includes reducing the lesion of endometriosis, in particular the lesion of uterine and / or uterine horn endometriosis. The treatment also includes chronic pelvic pain relief or vaginal hyperalgesia relief. It may also include a reduction in colorectal hyperalgesia developed in endometriosis, an improvement in bladder dysfunction developed in endometriosis, a reduction in skin sensitivity to enhanced thermal and / or mechanical stimulation developed in endometriosis, an improvement in overall health of altered endometriosis.

Description

[Technical Field]

[0001] This invention relates to medicines and methods for treating endometriosis, allowing for the reduction or disappearance of endometriotic lesions, and more specifically, combining beneficial therapeutic effects on endometriosis and markers of endometriotic lesions, as well as on associated pain (including vaginal hyperalgesia) and other concomitant conditions. [Background Technology]

[0002] Endometriosis is a chronic inflammatory gynecological disease affecting approximately 10% of women of reproductive age, causing nearly $80 billion in economic burden annually in the United States alone. Chronic pelvic pain (CPP) is the most debilitating symptom experienced by women with endometriosis. Furthermore, women with endometriosis are often diagnosed with visceral complications, including irritable bowel syndrome (IBS) and overactive bladder syndrome (OAB). Current research on the underlying mechanisms of endometriosis pain primarily focuses on the endometriotic lesions and their inflammatory environment as the source of CPP. However, surgical removal of endometriotic lesions does not reliably relieve pain, and adequate treatment remains lacking.

[0003] According to the World Health Organization, endometriosis is a disease characterized by the presence of tissue resembling the uterine lining outside the uterus. It causes a chronic inflammatory response that can lead to scarring (adhesions, fibrosis) in the pelvis and other parts of the body. Several types of lesions have been described, such as superficial endometriosis, primarily found on the pelvic peritoneum; cystic ovarian endometriosis (endometriotic cysts), found in the ovaries; and deep endometriosis, found in the rectovaginal septum, bladder, and intestines. In rare cases, endometriosis can also be found outside the pelvis. Symptoms associated with endometriosis vary and may include combinations of the following: dysmenorrhea, chronic pelvic pain, pain during and / or after intercourse, painful bowel movements, painful urination, fatigue, depression or anxiety, bloating, and nausea.

[0004] Currently, there is no known cure for endometriosis. Treatments typically aim to control symptoms, which is clearly insufficient.

[0005] Therefore, there is a need for new treatment methods that can reduce, limit, or eliminate lesions caused by endometriosis, and preferably, that can also reduce hyperalgesia or pain associated with endometriosis, as well as possible other complications or symptoms of comorbidities. [Summary of the Invention]

[0006] NP137 (a monoclonal antibody (mAb) against axonal guidance factor (netrin)-1 developed by Patrick Mehlen

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

[15] and is currently in a phase II clinical trial for advanced cancer (https: / / clinicaltrials.gov / ct2 / show / NCT02977195). Here it is shown for the first time that NP137 has an unusual and surprising ability in vivo to both reduce the development of endometriosis lesions and reduce chronic pelvic pain associated with endometriosis by alleviating vaginal hyperalgesia. Compelling data were obtained in two well-characterized mouse models of endometriosis that exhibited symptoms similar to those observed in women with endometriosis (i.e., the development of endometrial lesions and widespread chronic pelvic pain).

[0007] In one aspect, the present invention relates to the use of an anti-axon guide factor-1 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing an anti-axon guide factor-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable vehicle thereof, for the treatment of endometriosis, said treatment comprising reducing endometriotic lesions, particularly endometriotic lesions in the uterus and / or uterine horns.

[0008] In one embodiment, the anti-axon guidance factor-1 antibody and its antigen-binding fragment specifically bind to an axon guidance factor-1 epitope or binding region having the amino acid sequence SEQ ID NO: 3 or 33.

[0009] In another embodiment, the anti-axon guide factor-1 antibody and its antigen-binding fragment, as defined below, have a publicly disclosed set of 6 CDRs, or publicly disclosed specific VH and VL regions.

[0010] In a preferred embodiment, the anti-axon guide factor-1 antibody and antigen-binding fragment include the VH and VL pair of any one of the humanized monoclonal antibodies HUM01 to 10.

[0011] In a preferred embodiment, the anti-axon guide factor-1 antibody is NP137.

[0012] In a further aspect, the treatment includes reducing endometriotic lesions developing within the peritoneal cavity (i.e., around the uterus and / or uterine horns) and relieving chronic pelvic pain associated with endometriosis.

[0013] In particular, the treatment includes a reduction in endometriotic lesions and / or hemorrhagic lesions. Imaging allows for the observation, measurement, and consideration of the reduction in the total volume of lesions and / or the volume of hemorrhagic lesions. We consider a reduction to be significant when the total volume of lesions is reduced by more than 20%. In embodiments, the antibodies of the present invention allow for a reduction in lesions of more than 20, 30, 40, or 50%. Imaging can be performed using imaging methods for endometriosis imaging, such as transvaginal ultrasound, magnetic resonance imaging (MRI, see

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

[0014] Furthermore, NP137 treatment can improve vaginal hyperalgesia developed in endometriosis, as assessed in our model by electromyographic (EMG) signal analysis of visceral motor response (VMR) induced by vaginal dilation (VD), as described in Section 4. In women, we used the VAS (Visual Analogue Scale). Treatment is generally considered to have some degree of effectiveness in painful endometriosis when the pain scale shows a reduction of at least 2 VAS points (out of 10). In embodiments, the antibodies of the present invention allow for a reduction of at least 2, 3, or 4 VAS points. For more details on VAS, see

[10] .

[0015] In a further aspect, the treatment may further include one or more of the following additional technical effects or therapeutic functions:

[0016] • Reduced colorectal hyperalgesia that develops in endometriosis

[0017] • Improvement (75%) of bladder dysfunction developed in endometriosis.

[0018] • The decreased skin sensitivity to heat stimuli that develops in endometriosis

[0019] • The decreased skin sensitivity to mechanical stimulation that develops in endometriosis

[0020] • Improvement in overall health due to endometriosis changes.

[0021] In one aspect, the present invention relates to a method for treating endometriosis in women in need, comprising administering to the woman an effective amount of an anti-axon guide factor-1 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition containing an anti-axon guide factor-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable vehicle.

[0022] The method includes reducing endometriosis lesions, particularly endometriosis lesions in the uterus and / or uterine horns.

[0023] In a further aspect, the method includes reducing endometriosis lesions, particularly endometriosis lesions in the uterus and / or uterine horns, and relieving chronic pelvic pain or vaginal hyperalgesia.

[0024] In a further aspect, the method also includes one or more of the following additional effects:

[0025] • Reduced colorectal hyperalgesia developed in endometriosis.

[0026] • Improvement of bladder dysfunction that develops in endometriosis.

[0027] • Reduced skin sensitivity to heat stimulation that develops in endometriosis.

[0028] • Reduced skin sensitivity to mechanical stimulation, which develops in endometriosis.

[0029] • Improvement in overall health due to endometriosis changes.

[0030] The percentage reduction or improvement of the effects disclosed above also applies here.

[0031] In another aspect, the present invention relates to the use of the anti-axon guide factor-1 antibody or its antigen-binding fragment disclosed herein in the manufacture of a medicament for treating endometriosis (preferably endometriosis and pain as disclosed herein). [Detailed Description of the Invention]

[0033] The antibody can be a polyclonal or monoclonal antibody (anti-axon guide factor-1 antibody or antibody that binds to axon guide factor-1), especially human axon guide factor-1.

[0034] Anti-axon guide factor-1 polyclonal antibodies, among other things, can be obtained by immunizing animals (such as rabbits, mice, etc.) with a selected amino acid sequence, collecting the antibody, and then removing the antiserum obtained on, for example, an immunosorbent containing a receptor, according to methods known to those skilled in the art.

[0035] The amino acid sequence of axon guidance factor-1 is shown in SEQ ID NO: 1. Axon guidance factor-1 can be used in whole or in part to generate polyclonal or monoclonal antibodies.

[0036] Typically, monoclonal antibodies can be classified according to... The conventional methods of lymphocyte fusion and hybridoma culture described by Milstein are used to obtain these antibodies (Nature, 1975, 256(5517): 495-7). Other methods for preparing monoclonal antibodies are also known (Harlow et al., ed., 1988 "Antibodies: a laboratory manual"). Monoclonal antibodies can be prepared by immunizing mammals (e.g., mice, rats, rabbits, and even humans) and using lymphocyte fusion techniques that induce hybridomas. (and Milstein, 1975). Alternative techniques exist to this conventional approach. For example, monoclonal antibodies can be produced by expressing nucleic acids from a hybridoma clone. Antibodies can also be produced by introducing the antibody's cDNA into a vector, typically a filamentous phage, on which a gene library V is displayed (e.g., fUSE5 for E. coli, Scott JK, Smith GPScience 1990; 249:386-390). Protocols for constructing these antibody libraries are described in JD Marks et al., J. Mol. Biol., 222 (1991), p. 581. According to these methods, monoclonal antibodies can be produced using cDNA corresponding to full-length axon guidance factor-1 with a signal sequence (SEQ ID NO: 2) or a suitable fragment thereof.

[0037] Anti-axon guidance factor-1 monoclonal antibodies (mAbs) can be murine, chimeric, humanized, or fully human monoclonal antibodies. The fragment can be any type of mAb fragment that substantially retains the ability of the entire antibody to bind to axon guidance factor-1; for example, it can be Fab or F(ab')2. Specifically, the monoclonal antibody is one disclosed in WO2015 / 104360 or US10,494,427, which are incorporated herein by reference and disclose useful murine, chimeric, and humanized monoclonal antibodies, as well as methods for preparing the various mAbs described herein (e.g., HUM01–10, etc.). These are antibodies or fragments thereof that specifically bind to an axon guidance factor-1 epitope or a polypeptide having the amino acid sequence SEQ ID NO: 3 or 33, or a variant thereof.

[0038] The antibodies useful in this invention can be defined by their CDRs. Specifically, these CDRs are derived from the mouse antibody 4C11 disclosed in WO2015 / 104360 or US10,494,427, which specifically binds to a polypeptide having the amino acid sequence SEQ ID NO: 3 or 33. Preferably, the antibody is a monoclonal antibody or its antigen-binding fragment, comprising:

[0039] A variable structural domain VH, which contains:

[0040] H-CDR1, which has the sequence shown in SEQ ID NO: 5;

[0041] H-CDR2, which has the sequence shown in SEQ ID NO: 6;

[0042] H-CDR3, which has the sequence shown in SEQ ID NO: 7;

[0043] A variable domain VL, which contains:

[0044] • L-CDR1, which has the sequence shown in SEQ ID NO: 8;

[0045] L-CDR2, which has the sequence YAS;

[0046] • L-CDR3, which has the sequence shown in SEQ ID NO: 9; or

[0047] A variable structural domain VH, which contains:

[0048] H-CDR1, which has the sequence shown in SEQ ID NO: 28;

[0049] • H-CDR2, which has the sequence shown in SEQ ID NO: 29;

[0050] • H-CDR3, which has the sequence shown in SEQ ID NO: 30;

[0051] A variable domain VL, which contains:

[0052] • L-CDR1, which has the sequence shown in SEQ ID NO: 31;

[0053] • L-CDR2, which has the sequence shown in SEQ ID NO: 32;

[0054] ·L-CDR3, which has the sequence shown in SEQ ID NO: 9.

[0055] In a first series of embodiments, the antibody of the present invention comprises the amino acid sequence SEQ ID NO: 10, 11, 12 or 13. Typically, it comprises the VH of sequence SEQ ID NO: 10 and the VL of sequence SEQ ID NO: 11, or the heavy chain of sequence SEQ ID NO: 12 and the light chain of sequence SEQ ID NO: 13.

[0056] In the second series of embodiments, the antibody is chimeric. Preferably, it comprises VH of sequence SEQ ID NO: 27 and VL of sequence SEQ ID NO: 19.

[0057] In the third series of embodiments, the antibody is humanized. Preferably, it comprises an amino acid sequence selected from the SEQ ID NO: 14-18 (VL) group and / or from the SEQ ID NO: 20-26 (VH) group. Typically, the antibody is humanized and comprises a VH having an amino acid sequence selected from the SEQ ID NO: 14-18 group and a VL having an amino acid sequence selected from the SEQ ID NO: 20-26 group.

[0058] The antibody preferably comprises a monoclonal antibody (mAb) or an antigen-binding fragment thereof, wherein the mAb or fragment thereof specifically binds to axon guidance factor-1. The mAb can be a mouse, chimeric, humanized, or fully human monoclonal antibody. The fragment can be any type of mAb fragment that substantially retains the ability of the entire antibody to bind to axon guidance factor-1; for example, it can be Fab or F(ab')2.

[0059] The specific embodiments disclosed in this prior literature and applicable herein are the antibodies listed in Table 1. The first antibody listed is a chimeric 4C11 antibody, including mouse VH and VL of mouse 4C11 antibody. HUM00 listed in Table 1 corresponds to the grafting of mouse 4C11 CDR into human IgG1. Ten humanized mAbs HUM01 to HUM10 correspond to humanized mAbs derived from HUM00, having the same CDR but specifically modified in the FR region of human IgG. HUM03 is also disclosed as NP137 and is currently undergoing clinical trials. The sequence of human IgG1 CH is from Genbank AEL33691.1 (modified with R97K). The sequence of human IgG1 CL(κ) is from Genbank CAC20459.1. Other allotypes may also be used. The specific binding of all these mAbs, mouse, chimeric and humanized HUM01–HUM10, Fab fragments and F(ab')2 fragments to axon guidance factor-1 and their ability to inhibit the binding of axon guidance factor-1 to its receptor UNC5B has been demonstrated in US 10,494,427 (Example 3).

[0060] In particular, these antibodies specifically bind to a polypeptide having the amino acid sequence SEQ ID NO: 33.

[0061] Table 1

[0062] VH SEQ ID NO: constant heavy chain L SEQ ID NO: constant light chain 4C11 10 Human IgG1 11 Human IgG1 HUM00 27 Human IgG1 19 Human IgG1 HUM01 20 Human IgG1 4 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 7 Human IgG1 HUM08 22 Human IgG1 17 Human IgG1 HUM09 25 Human IgG1 18 Human IgG1 HUM10 21 Human IgG1 16 Human IgG1

[0063] Preferably, the antibody is a monoclonal antibody or its antigen-binding fragment, comprising a VH and VL sequence pair 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 its antigen-binding fragment, comprising a VH and VL sequence pair SEQ ID NO: 22 and 16.

[0064] Anti-axon guidance factor-1 antibodies may further include human IgG1 constant heavy chain (CH) and / or human IgG1 constant light chain (CL), particularly the human κ constant domain.

[0065] In one embodiment, the human IgG1 CH sequence is from Genbank AEL33691.1 (R97K modified). The human IgG1 CL(κ) sequence is from Genbank CAC20459.1. In one embodiment, the mAb is NP137 (AB_2811180 in the antibody registry) and contains SEQ ID NO: 22 and 16 as VH and VL sequences, respectively, as well as those specific to IgG1 CH and CL.

[0066] As used herein, the term "antigen-binding fragment" (or "antibody-binding portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to axon guidance factor-1. Antibody fragments may include, for example, Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing a CDR, or isolated CDRs. In one aspect, the fragment contains the VH and VL sequences of an antibody selected from HUM00 to HUM10.

[0067] As potential anti-axon guidance factor-1 antibodies, other antibodies developed against human or animal axon guidance factor-1, especially monoclonal antibodies or their antigen-binding fragments, can be cited. Axon guidance factor-1 is highly homologous across species. Examples include: Abcam antibodies ab126729, ab122903, ab201324, ab39370; AF1109, AF6419, AF128.

[0068] In one embodiment, the antibody or antigen-binding fragment thereof used in the purpose or method of the invention is administered as a single dose, or as a second or more subsequent doses of the antibody or antigen-binding fragment after an initial dose, wherein the subsequent doses are spaced 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 twelve weeks; or at least fourteen weeks apart. The dosage may vary depending on the age and weight of the subject to be administered, the target disease, the condition, the route of administration, etc. When the antibody of the invention is used to treat pain in adult patients, intravenous, subcutaneous, or intraperitoneal administration of the antibody of the invention is advantageous. A single dose or subsequent doses may comprise at least 0.1 or 1 mg / kg body weight of the antibody or a fragment thereof, for example, from about 1 mg / kg body weight to about 100 mg / kg body weight, particularly from about 10 mg / kg body weight to about 60 mg / kg body weight in the subject. The frequency and duration of treatment may be adjusted according to the severity of endometriosis, such as the number and extent of lesions, and the degree of pain and / or comorbidities.

[0069] [Definition, further embodiments, variations and alternatives of this invention]

[0070] Throughout this immediate application, the term "comprising" should be interpreted as including all specifically mentioned functions as well as optional, additional, and unspecified functions. As used herein, the use of the term "comprising" also discloses embodiments in which no other features exist besides the specifically mentioned features (i.e., "consisting of," "contributing to").

[0071] In the context of this invention, the term “treating” or “treatment” as used herein means achieving a reduction or disappearance of lesions, and preferably, reducing or alleviating endometriosis-related pain and / or endometriosis-related complications, as disclosed herein.

[0072] The "therapeutic effective dose" of the antibody of this invention refers to a sufficient amount to treat the pain with a reasonable benefit / risk ratio suitable for any drug treatment. However, it is understood that the total daily dosage of the antibody will be determined by the attending physician within reasonable medical judgment. The specific therapeutic effective dose level for any particular 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 ingredients employed, the woman's age, weight, general health condition, and diet; the timing, route of administration, and excretion rate of the specific antibody used; the duration of treatment; the drugs used in combination with or concurrently with the specific antibody used; and similar factors well known in the medical field.

[0073] "Pharmaceutical-acceptable" or "pharmaceutical-admissible" means a molecular entity or composition that, when administered to mammals (especially humans) under appropriate conditions, will not produce adverse, allergic, or other adverse reactions. A pharmaceutically acceptable vehicle (or carrier or excipient) means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation adjuvant.

[0074] The form of the pharmaceutical composition, including the antibodies of the present invention and the route of administration, naturally depends on the condition to be treated, the severity of the disease, the patient's age, weight, and sex, etc.

[0075] The antibodies of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. In a specific embodiment, the antibodies of the present invention are administered intravenously.

[0076] In particular, pharmaceutical compositions including the antibodies of the present invention may contain a vehicle that is pharmaceutically acceptable for injectable formulations. These may be particularly isotonic, sterile saline solutions (sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dry, particularly lyophilized compositions, with the addition of sterile water or physiological saline as appropriate, to form injectable solutions.

[0077] Suitable drug forms for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to a degree that facilitates injection. It must be stable under the conditions of manufacture and storage and must be preserved against contamination by microorganisms such as bacteria and fungi.

[0078] The vehicle or carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using a coating (such as lecithin), by maintaining the desired particle size in the dispersed state, and by using surfactants, stabilizers, cryoprotectants, or antioxidants. Antimicrobial activity can be achieved through antibacterial and antifungal agents. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred.

[0079] Sterile injection solutions are prepared by incorporating the active compound in the desired amount with several other components listed above into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and the other desired components mentioned above. For sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying techniques to produce powders of the active ingredient and any additional desired components from a previously sterile filtered solution.

[0080] After preparation, the solution will be administered in a manner compatible with the dosage formulation and at a therapeutically effective amount. This preparation is readily administered in various dosage forms, such as the aforementioned injection type, but drug-release capsules may also be used.

[0081] For example, for parenteral administration in aqueous solutions, an appropriate buffer solution should be used if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, those skilled in the art will know, based on this disclosure, the sterile aqueous media that can be used. For example, a dose can be dissolved in 1 mL of isotonic NaCl solution and then added to 1000 mL of subcutaneous injection or injected into the recommended infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). The dose will inevitably vary depending on the condition of the treated subject. In any case, the person responsible for administration will determine the dose appropriate for the individual subject.

[0082] Table 2: Sequence Description

[0083]

[0084]

[0085]

[0086]

[0087] The CDRs under IMGT are highlighted in bold in Table 1 as appropriate. [Definitions, Further Embodiments, Variations, and Alternatives of the Invention]

[0088] As used herein, a sequence that is “at least 85% identical to a reference sequence” means a sequence that has 85% or higher, particularly 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 a reference sequence over its entire length.

[0089] The percentage of “sequence identity” can be determined by comparing two sequences, performing an optimal alignment on a comparison window. This window includes portions of the polypeptide sequence containing additions or deletions (i.e., vacancies) compared to a reference sequence (excluding additions or deletions) to achieve the best alignment between the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same amino acid residues appear, resulting in a number of matching positions. This number is then divided by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the percentage of sequence identity. Optimal alignment of the sequences used for comparison is performed using global pairwise alignment, for example, using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443. For example, the percentage of sequence identity can be easily determined using the program Needle (containing a BLOSUM62 matrix and the following parameters: gap-open = 10, gap-extend = 0.5).

[0090] In the context of this invention, "conservative amino acid substitution" refers to an amino acid residue that is replaced by another amino acid residue of a side chain group having similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially alter the functional properties of a protein. Examples of amino acid groups with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy 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; (7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0091] Antibodies can be natural or conventional, consisting of two heavy chains linked together by disulfide bonds, each of which is connected to a light chain via a disulfide bond. There are two types of light chains, λ and κ. Five main heavy chain classes (or isotypes) determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine antigen binding recognition and specificity. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment, consisting of a variable portion of a light chain and a heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues from the hyperdenaturing or complementarity-determining region (CDR). Sometimes, residues from the non-hyperdenaturing or framework regions (FR) can affect the overall domain structure, thus influencing the binding site.

[0092] "Complementarity-determining regions" or "CDRs" refer to amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. Each of the light and heavy chains of immunoglobulins has three CDRs, named CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, a typical antibody-antigen binding site contains six CDRs, comprising CDR sets from the respective V regions of the heavy and light chains.

[0093] The "frame region" (FR) refers to the amino acid sequence between the CDRs, which are the relatively conserved variable regions of the light and heavy chains of immunoglobulins within a single species. Each immunoglobulin light and heavy chain has four FRs, named FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

[0094] As used in this article, "human frame region" is a frame region that is substantially identical (approximately 85%, or more, particularly 90%, 95%, 97%, 99%, or 100%) to the frame region of naturally occurring human antibodies.

[0095] In the context of this invention, the CDR / FR definition in the immunoglobulin light or heavy chain is determined based on the definition of Kabat or IMGT.

[0096] Residues in the antibody variable domain are typically numbered according to a system devised by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This specification uses this numbering system. Kabat residue names do not always directly correspond to the linear numbers of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat number, corresponding to shortening or insertion of structural components of the basic variable domain structure (whether framework or complementarity-determining regions (CDRs)). The correct Kabat residue number for a given antibody can be determined by comparing homologous residues in the antibody sequence to a "standard" Kabat numbered sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31–35B (H-CDR1), residues 50–65 (H-CDR2), and residues 95–102 (H-CDR3). According to the Kabat numbering system (http: / / www.bioinf.org.uk / abs / #cdrdef), the CDRs of the variable domains in the light chain are located at residues 24–34 (L-CDR1), residues 50–56 (L-CDR2), and residues 89–97 (L-CDR3).

[0097] In the context of this invention, the amino acid residues of the antibody of this invention may be numbered according to the IMGT numbering system. The unique IMGT number has been defined for comparing variable domains of any 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., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains” Dev. Comp. Immunol., 27, 55-77 (2003).). In the unique IMGT numbering, conserved amino acids always occupy the same position, for example, cysteine ​​23, tryptophan 41, hydrophobic amino acid 89, cysteine ​​104, phenylalanine, or tryptophan 118. The unique IMGT numbering provides standard delimitations for the framework region (FR1-IMGT: positions 1–26, FR2-IMGT: 39–55, FR3-IMGT: 66–104, and FR4-IMGT: 118–128) and the complementarity-determining region (CDR1-IMGT: 27–38, CDR2-IMGT: 56–65, and CDR3-IMGT: 105–117). If the CDR3-IMGT length is less than 13 amino acids, vacancies are generated from the top of the ring in the following order: 111, 112, 110, 113, 109, 114, etc.If the CDR3-IMGT length exceeds 13 amino acids, additional positions are generated between positions 111 and 112 at the top of the CDR3-IMGT ring in the following order: 112.1, 111.1, 112.2, 111.2, 112.3, 111.3, etc. (http: / / www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html).

[0098] As used herein, the term “antibody” refers to conventional antibodies and their fragments, as well as single-domain antibodies and their fragments, particularly variable heavy chains of single-domain antibodies, and chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.

[0099] As used herein, antibodies or immunoglobulins also include the recently described “single-domain antibodies,” which are antibodies whose complementation-determining regions are part of a single-domain polypeptide. Examples of single-domain antibodies include heavy-chain antibodies, naturally occurring antibodies that do not contain a light chain, 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 mice, humans, camels, llamas, goats, rabbits, and cattle. Single-domain antibodies may be naturally occurring single-domain antibodies, referred to as light-chain-free heavy-chain antibodies. In particular, camel species, such as camels, dromedary camels, llamas, alpacas, and guanacos, produce naturally occurring light-chain-free heavy-chain antibodies. Camel heavy-chain antibodies also lack the CH1 domain.

[0100] The variable heavy chains of these light-chain-less, single-domain antibodies are referred to in the art as “VHH” or “nanobodies.” Similar to the conventional VH domain, a VHH contains four free radicals (FRs) and three core radicals (CDRs). Nanobodies offer advantages over conventional antibodies: they are approximately ten times smaller than IgG molecules, allowing for the generation of appropriately folded functional nanobodies through in vitro expression in high yields. Furthermore, nanobodies are highly stable and resistant to protease activity. The properties and production of nanobodies have been reviewed by Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22.

[0101] The term "monoclonal antibody" or "mAb" as used in this article refers to an antibody molecule composed of a single amino acid against a specific antigen and should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced from a single clone of B cells or hybridomas, but they can also be recombinant, i.e., produced through protein engineering.

[0102] A "fragment" of a (conventional) antibody includes a portion of the complete antibody, particularly the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, biantibodies, and bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single-domain antibodies, such as heavy chain antibodies or VHH.

[0103] The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 Da and antigen-binding activity, wherein in the fragment obtained by treating IgG with the protease papain, the N-terminal side of the H chain and approximately half of the entire L chain are linked together by disulfide bonds.

[0104] The term "F(ab')2" refers to an antibody fragment with a molecular weight of approximately 100,000 Da and antigen-binding activity. In fragments obtained by treating IgG with the protease pepsin, this antibody fragment is slightly larger than the Fab bound by disulfide bonds through the hinge region.

[0105] A single-chain Fv (“scFv”) polypeptide is a covalently linked VH::VL heterodimer, typically expressed by gene fusion comprising VH and VL encoding genes linked via peptide-coding linkers. The human scFv fragments of this invention contain a CDR that maintains an appropriate conformation, particularly through the use of gene recombination techniques. Bivalent and multivalent antibody fragments can be spontaneously formed by binding to a monovalent scFv, or generated by peptide linker coupling to a monovalent scFv (e.g., a bivalent sc(Fv)2).

[0106] “dsFv” is a VH::VL heterodimer stabilized by disulfide bonds.

[0107] “(dsFv)2” represents two dsFvs coupled through a peptide linker.

[0108] The term "bispecific antibody" or "BsAb" refers to an antibody that binds the antigen-binding sites of two antibodies within a single molecule. Therefore, a BsAb can bind to two different antigens simultaneously. Genetic engineering is increasingly used to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described in EP 2 050764A1, for example.

[0109] The term "multispecific antibody" refers to an antibody that binds the antigen-binding sites of two or more antibodies within a single molecule.

[0110] The term "dual antibody" refers to a small antibody fragment with two antigen-binding sites. These fragments contain a heavy chain variable domain (VH) that is linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with a complementary domain of the other chain, creating two antigen-binding sites.

[0111] In a particular implementation, the epitope-binding fragment is selected from the group consisting of: Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, double-chain antibody, and VHH.

[0112] The term "chimeric antibody" as used in this article refers to an antibody in which the constant region or a portion thereof is altered, replaced, or exchanged, so that the variable region is linked to the constant region of a different species, or belongs to another antibody class or subclass. "Chimeric antibody" also refers to an antibody in which the variable region or a portion thereof is altered, replaced, or exchanged, so that the constant region is linked to the variable region of a different species, or belongs to another antibody class or subclass.

[0113] The term "humanized antibody" refers to an antibody that is originally wholly or partially non-human in origin and has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize human immune responses. The constant domains of humanized antibodies are, most often, human CH and CL domains. In embodiments, humanized antibodies have human constant domains. As used herein, the term "humanized antibody" refers to a chimeric antibody containing a minimal sequence (e.g., CDR) derived from a non-human immunoglobulin.

[0114] The term "antibody" is used to encompass all these types of antibodies, fragments, or combinations thereof.

[0115] The goal of humanization is to reduce the immunogenicity of xenobiotic antibodies (e.g., mouse antibodies) when introduced into the human body, while maintaining the antibody's complete antigen-binding affinity and specificity. Humanized antibodies, or antibodies adapted to be non-rejected by other mammals, can be produced using a variety of techniques, such as surface remodeling and CDR transplantation. As used in this paper, surface remodeling techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to alter the non-CDR surface of the antibody's variable region, making it similar to the surface of a known antibody in the target host.

[0116] Antibodies can be humanized using a variety of other techniques, including CDR transplantation (EP0239400; WO91 / 09967; US Patent Nos. 5,530,101 and 5,585,089), surface finishing or remodeling (EP0592106; EP0519596; Padlan (1991) Molecular Immunology 28(4 / 5):489-498; Studnicka et al. (1994) Protein Engineering 7(6):805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci USA91:969-973) and chain tampering (US Patent No. 5,565,332). Human antibodies can be prepared using a variety of methods known in the art, including phage display methods. See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and international patent applications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735 and WO91 / 10741.

[0117] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen (e.g., axon guidance factor-1) while having relatively low detectable reactivity with non-axon guidance factor-1 proteins or structures (e.g., other proteins presented on cancer cells or other cell types). Specificity can be relatively determined by binding or competitive binding assays, for example, using a Biacore instrument, as described elsewhere herein. Specificity can be expressed, for example, as the affinity / affinity for binding to a specific antigen compared to the affinity / affinity for nonspecific binding to other unrelated molecules (in this case, axon guidance factor-1) in ratios of approximately 10:1, approximately 20:1, approximately 50:1, approximately 100:1, approximately 10,000:1, or higher.

[0118] The term “affinity” as used in this paper refers to the strength of the binding between an antibody and an epitope. The affinity of an antibody is given by the dissociation constant Kd, which is defined as [1]×[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 as 1 / Kd. The preferred method for determining mAb affinity is found in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY, (1988); Coligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. A preferred and standard method known in the art for determining mAb affinity is the use of a Biacore instrument.

[0119] The invention will now be described in more detail with reference to the accompanying drawings and the following non-limiting experiments. [Attached Image Description]

[0120] Figure 1 This diagram shows the timeline of the different interventions conducted in this study.

[0121] (A) Ovariectomy was performed on mice one week (-1 week) before surgical induction of uterine horn fragmentation (Endo) or suturing only (Sham) on day 0 (d0). All mice were injected intraperitoneally weekly with estradiol benzoate (red inverted triangle) to maintain stable circulating estrogen levels throughout the study. Treatment began 4 weeks post-operative induction and mice were grouped as follows: Endo mice treated with NP137 (Endo + NP137 (N=15)) or Endo mice treated with the control antibody NP001 (Endo + control Ab (N=16)) and Sham mice treated with control Ab NP001 (Sham + control Ab (N=16)). Intraperitoneal treatment was maintained every two weeks throughout the study. In vivo pain assessments were conducted between weeks 8 and 10 post-operative induction, comparing Sham (control Ab) and Endo (control Ab and NP137) (shaded box). Tissue samples were collected from all mice following a final behavioral assessment (diamond). The total number of mice used in this study was N=16 Sham (control Ab), N=16 Endo (control Ab), and N=15 Endo (NP137).

[0122] (B) Ovariectomy was performed on mice one week prior to day 0 (d0) inoculation with uterine horn fragments (Endo) or saline-only inoculation (Sham). Estradiol benzoate (inverted triangle) was injected intraperitoneally into all mice weekly to maintain stable circulating estrogen levels throughout the study. Treatment began 4 weeks post-operative induction and mice were grouped as follows: Endo mice treated with NP137 (Endo + NP137) or Endo mice treated with the control antibody NP001 (Endo + control Ab (N=8)) and Sham mice treated with control Ab NP001 (Sham + control Ab (N=4)). Intraperitoneal treatment was maintained every two weeks throughout the study. In vivo pain assessments were conducted between weeks 8 and 10 post-operative induction, comparing Sham (control Ab) and Endo (control Ab and NP137) (shaded box). Tissue samples were collected from all mice after behavioral assessments using a hot plate test (diamond). The total number of mice used was N=4 Sham (control Ab), N=8 Endo (control Ab), and N=7 Endo (NP137).

[0123] Figure 2 NP137 treatment reduces the size of endometriosis lesions.

[0124] (A1) Representative images of the reproductive tract of the following mice 10 weeks after surgical induction: Sham mice treated with control Ab (left box, sutured only at the uterine horn and parauterus (arrows and arrowheads indicate the location)), and Endo mice treated with control Ab (middle box) or NP137 (right box) (endometrial fragments attached to the uterine horn and parauterus (arrows indicate the location)).

[0125] (A2) Representative images of anatomical and cleaned endometriosis lesions taken from the uterus and uterine horns of the following two types of Endo mice: Endo mice treated with control Ab (4 mice, left box) and Endo mice treated with NP137 (3 mice, right box). Scale bar represents 5 mm.

[0126] (B) Group data showed that, compared with the control Ab, the mean lesion weight was significantly reduced 10 weeks after Endo induction following long-term treatment with NP137. The group data are shown as (B1) mean lesion weight (g) for all lesions per animal, (B2) mean lesion weight (g) for lesions located in the uterus, and (B3) mean lesion weight (g) for lesions located in the uterine horn.

[0127] (C) Group data showed that, compared with the control Ab, the mean maximum lesion diameter was significantly reduced 10 weeks after Endo induction following long-term NP137 treatment. Group data are presented as (C1) mean maximum lesion diameter (mm) for all lesions per animal, (C2) mean maximum lesion diameter (mm) for lesions located in the uterus, and (C3) mean maximum lesion diameter (mm) for lesions located in the uterine horn. Data are expressed as mean ± SEM. NS, P > 0.05, ** P < 0.01, *** P < 0.001. Mann-Whitney nonparametric test was used for two groups with unequal variances. Each treatment group had N = 9–11 mice and N = 29–35 lesions.

[0128] Figure 3 NP137 treatment reduced vaginal hyperalgesia developed in mice with endometriosis.

[0129] (A) Grouped data showed that, compared with Sham mice treated with control Ab, Endo mice treated with control Ab exhibited increased visceral motor response (VMR) to vaginal dilation (VD) at 20 mmHg–70 mmHg. In mice treated with NP137, Endo mice showed a significantly reduced increased response to harmless dilation pressure. ^ indicates Sham control Ab versus Endo control Ab, * indicates Endo control Ab versus Endo NP137. Data are presented as mean ± SEM. * or ^P < 0.05, ** or ^^P < 0.01, **** or ^^^^P < 0.0001, and LSD post-hoc tests were performed after generalized estimation equations.

[0130] Data for groups (B–D) are represented as the total area under the curve (AUC) of VMR for diastolic pressure against VD: (B) full diastolic pressure (20–80 mmHg), which was further divided into (C) low (20–40 mmHg) diastolic pressure and (D) high (60–80 mmHg) diastolic pressure, indicating that the elevated response observed in Endo mice treated with control Ab was normalized by treatment with NP137 compared with Sham mice treated with control Ab. Data are expressed as mean ± SEM, NS = not significant, *P < 0.05 and **P < 0.01. Conventional one-way ANOVA and post-hoc Sidak multiple comparisons were used for homogeneous variance groups, with N = 12–14 mice per treatment group. Each point represents the AUC from an individual animal.

[0131] Figure 4 NP137 treatment reduced colonic hyperalgesia developed in mice with endometriosis.

[0132] (A) Grouped data showed that Endo mice (yellow symbols) exhibited increased visceral motor response (VMR) to colorectal dilatation (CRD) compared to Sham mice (blue symbols) treated with control Ab. In mice treated with NP137 (green symbols), Endo mice showed a significantly reduced response to elevated CRD levels of 20 mmHg–60 mmHg. ^ indicates Sham control Ab versus Endo control Ab, and * indicates Endo control Ab versus Endo NP137. Data are presented as mean ± SEM. * or ^P < 0.05, *** or ^^^P < 0.001, **** or ^^^^P < 0.0001, and LSD post-hoc tests were performed after applying the generalized estimation equation.

[0133] Groups (B–D) are represented as the total area under the curve (AUC) of diastolic pressure against CRD: (B) full diastolic pressure (20–80 mmHg), which was further divided into (C) low (20–40 mmHg) diastolic pressure and (D) high (50–80 mmHg) diastolic pressure, indicating that the elevated response observed in Endo mice treated with control Ab was normalized by treatment with NP137 compared with Sham mice treated with control Ab. Data are expressed as mean ± SEM, NS = not significant, **P < 0.01, **P < 0.001, and ****P < 0.0001. Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances, with N = 12–14 mice per treatment group. Each point represents the AUC from an individual animal.

[0134] Figure 5 Bladder dysfunction developed in mice with torsion endometriosis treated with NP137.

[0135] (A) Grouped data of total urinary spot counts in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137 showed that the total urinary spot count produced by Endo mice was normalized by treatment with NP137.

[0136] (B) Group data showed that urination patterns (total urinary spots) were categorized as small (100–1,000 dpi), medium (1,001–100,000 dpi), or large (>100,001 dpi) urinary spots in Sham mice treated with control Ab (blue symbols), Endo mice treated with control Ab (yellow symbols), and Endo mice treated with NP137 (green symbols). The data showed that the significant increase in small spots produced by Endo mice was normalized by NP137 treatment. Data are expressed as mean ± SEM, NS = not significant, *P < 0.05 and **P < 0.01. Ordinary one-way ANOVA and Turkey's post-hoc multiple comparisons test were used for groups with homogeneous variances; or Kruskal-Wallis test and Dunn's multiple comparisons test were used for groups with unequal variances. N = 13–16 mice per treatment group. Each point represents one individual animal.

[0137] Figure 6 Mechanical and thermal skin hypersensitivity reactions developed in mice with torsion endometriosis treated with NP137.

[0138] (A) Grouped data showed the mechanical paw retraction threshold induced by stimulation of the hind paw (g) with an electronic von Frey (EvF) filament in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data showed that Endo mice exhibited enhanced sensitivity to mechanical stimulation compared to Sham mice treated with control Ab, which was normalized by treatment with NP137.

[0139] (B) Group data show the response time (in seconds) to a hot plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data showed that the increased sensitivity to harmful thermal stimuli exhibited by Endo mice compared to Sham mice treated with control Ab was absent in Endo mice treated with NP137. Data are expressed as mean ± SEM. NS = not significant, *P < 0.05, **P < 0.01, and ****P < 0.0001. Ordinary one-way ANOVA and Turkey's post-hoc multiple comparisons test were used for groups with homogeneous variances; or Kruskal-Wallis test and Dunn's multiple comparisons test were used for groups with unequal variances. N = 14–16 mice per treatment group. Each point represents one individual animal.

[0140] Figure 7 NP137 treatment improved signs of overall declining health in nesting observed in mice with endometriosis. Group data showed that nesting scores (1–5 points) in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137 normalized the reduced nesting behavior observed in Endo mice treated with control Ab with NP137 treatment. Data are presented as mean ± SEM. NS = not significant and *P < 0.05. Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances. N = 14–16 mice per treatment group. Each point represents one individual animal.

[0141] Figure 8 NP137 treatment tends to reverse the thermal skin hypersensitivity that develops in a mouse model of endometriosis.

[0142] Group data showed the response time (in seconds) to a hot plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data showed that Endo mice exhibited a trend toward increased sensitivity to harmful thermal stimuli compared to Sham mice treated with control Ab, a trend partially reversed in Endo mice treated with NP137. Data are expressed as mean ± SEM, NS = not significant. For groups with homogeneous variance, ordinary one-way ANOVA and Turkey's post-hoc test for multiple comparisons were used; or for groups with unequal variances, Kruskal-Wallis test and Dunn's multiple comparisons were used. N = 4–8 mice per treatment group. Each point represents one individual animal.

[0143] Figure 9 The improvement in overall health of Endo mice induced by NP137 was confirmed in a new animal cohort. Group data showed that nesting scores (1–5 points) in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137 normalized the reduced nesting behavior observed in Endo mice treated with control Ab with NP137 treatment. Data are presented as mean ± SEM. NS = not significant, *P < 0.05. Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances. N = 20–21 mice per treatment group. Each point represents one individual animal.

[0144] Figure 10 NP137 treatment tends to reduce the size and number of endometriosis lesions developed in mouse models of endometriosis syngeneia.

[0145] (A) Group data showed that, compared with the control Ab, the mean maximum lesion diameter was slightly reduced 10 weeks after Endo induction following long-term NP137 treatment.

[0146] (B) Group data showed that, compared with the control Ab, the average lesion size per animal was slightly reduced 10 weeks after Endo induction following long-term treatment with NP137.

[0147] (C) Group data showed that, compared with the control Ab, the number of lesions developed per animal was slightly reduced 10 weeks after Endo induction following long-term treatment with NP137.

[0148] Data are expressed as mean ± SEM. NS, P > 0.05. The Mann-Whitney nonparametric test was used for two groups with unequal variances, and the unpaired t-test was used for two groups with homogeneous variances. Each treatment group had N = 5 mice and n = 24–33 lesions.

[0149] Figure 11 The reduction in the size of endometriosis lesions induced by NP137 has been confirmed in a new animal cohort.

[0150] (A1) Representative images of the reproductive tract of the following mice 10 weeks after induction surgery: Sham mice treated with control Ab (top image, sutured only at the uterine horn and parauterus (arrow endmarks indicate location)), Endo mice treated with control Ab (middle image), or Endo mice treated with NP137 (bottom image) (endometrial fragments attached to the uterine horn and parauterus (arrow endmarks indicate location)).

[0151] (A2) Representative images of anatomical and cleaned endometriosis lesions taken from the uterus and uterine horn of the following two types of mice: Endo mice treated with control Ab (4 mice, top) and Endo mice treated with NP137 (4 mice, bottom).

[0152] (B) Group data showed that, compared with the control Ab, the mean lesion weight was significantly reduced 10 weeks after Endo induction following long-term treatment with NP137. Group data are shown as (B1) mean lesion weight (g) for all lesions per animal, (B2) mean lesion weight (g) for lesions located in the uterus, and (B3) mean lesion weight (g) for lesions located in the uterine horn.

[0153] (C) Group data showed that, compared with the control Ab, the mean maximum lesion diameter was significantly reduced 10 weeks after Endo induction following long-term NP137 treatment. Group data are presented as (C1) mean maximum lesion diameter (mm) for all lesions per animal, (C2) mean maximum lesion diameter (mm) for lesions located in the uterus, and (C3) mean maximum lesion diameter (mm) for lesions located in the uterine horn. Data are expressed as mean ± SEM. NS, P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001. For unequal variances between the two groups, the Mann-Whitney nonparametric test was used. Each treatment group had N = 12–16 mice and N = 44–53 lesions.

[0154] Figure 12 The reduction of NP137-induced thermal skin hypersensitivity was confirmed in a new animal cohort.

[0155] (A) Grouped data showed the mechanical paw retraction threshold (g) induced by electron von Frey (EvF) filament stimulation in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data showed that the enhanced sensitivity to mechanical stimulation exhibited by Endo mice was normalized by treatment with NP137 compared to Sham mice treated with control Ab.

[0156] (B) Group data showed the response time (in seconds) to the hot plate in Sham mice treated with control Ab, Endo mice treated with control Ab, and Endo mice treated with NP137. The data showed that the increased sensitivity to harmful thermal stimuli exhibited by Endo mice compared to Sham mice treated with control Ab was not present in Endo mice treated with NP137. Data are expressed as mean ± SEM. NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. For groups with homogeneous variance, ordinary one-way ANOVA and Turkey's post-hoc multiple comparisons test were used; or for groups with unequal variance, Kruskal-Wallis test and Dunn's multiple comparisons were used. N = 20–22 mice per treatment group.

Example

[0157] 【1. Animals】

[0158] The Animal Ethics Committee of the South Australian Institute of Health and Medical Research (SAHMRI) approved all experiments involving animals (ethics number SAM342). All animal experiments complied with relevant regulatory standards and ARRIVE guidelines. Six-week-old female C57BL / 6J mice were used and obtained from the in-house C57BL / 6J breeding program (Jax strain #000664; originally purchased from Jackson Laboratories (breeding farm MP14; Bar Harbor, ME; USA)) within SAHMRI's specific and opportunistic pathogen-free animal care facility. All female mice used in this study were virgins (never mated) and were housed without males since weaning. Mice were individually housed after surgery in coarse-fiber, dust-free aspen bedding ( The animals were housed in individually ventilated cages (IVCs) in a controlled humidity and temperature environment within the aviary, maintained at 22±1°C and a 12-hour light / 12-hour dark cycle. Mice were provided free of charge. JL rats and mice / Auto6F diet (Cat#5K52, St. Louis, MO; USA) and provided autoclaved reverse osmosis purified water.

[0159] [2. Surgery-induced mouse model of endometriosis]

[0160] For this study, we used a modified version of our previously established surgically induced autologous mouse model of endometriosis [9] (the entire contents of which are incorporated herein by reference). In this study, a total of four donor uterine horn fragments were surgically sutured adjacent to the female reproductive tract, two of which were located adjacent to the uterus (one on each side) and two attached to the uterine horns (one in each horn). See Figure 1 The diagram shows the timeline of the different interventions conducted in this study.

[0161] 2.1. Ovarian removal surgery

[0162] Seven days prior to surgical induction of endometriosis, female mice underwent ovariectomy to deplete endogenous steroid production. Briefly, mice were anesthetized with isoflurane (2.5% / 0.5L O2) and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before surgery. Under aseptic conditions, a small longitudinal paralumbar incision was made in the skin and dorsal abdominal muscles to expose the bilateral ovaries. The ovaries were removed, and the fallopian tube / ovarian artery and vein / ovarian ligament were severed by cauterization. The uterine horn was then returned to the abdominal cavity and clamped with a 9mm clip ( The superficial skin layer was closed by the system (FST). Ovarian-removed mice were allowed at least 5 days to recover before removal of the surgical clips. Since endometriosis is an estrogen-dependent disease, maintaining stable circulating estrogen levels and minimizing any differences associated with estrous cycle phases is important. Taking these factors into account, all mice were intraperitoneally injected with 100 μg / kg estradiol benzoate (Progynon-B) once weekly throughout the study after ovariectomy.

[0163] 2.2. Endometriosis and Sham Surgery

[0164] Seven days after ovariectomy, endometriosis was surgically induced in experimental mice using uterine horn tissue collected from donor mice. To collect donor tissue, the uterine horn was excised and clamped into a dissecting dish (Sigma-Aldrich, St. Louis, MO) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) in ice-cold sterile phosphate-buffered saline (PBS). The uterine horn was longitudinally dissected, and 4 mm × 2 mm tissue sections were collected using a 2 mm biopsy punch (Kai medical, KAI00010). Recipient mice were then anesthetized with isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before surgery. Under aseptic conditions, a small midline abdominal incision was made to expose the peritoneum, and 2 mm circular fragments of donor uterine horn tissue were sutured to the parametrial region (2 fragments) and the parametrial region (1 fragment per horn) using 6.0 Prolene sutures. For the Sham procedure, 6.0 Prolene sutures of similar length were connected to the same surgical site as in the Endo procedure, but without any tissue. Subsequently, the abdominal muscles were sutured and clipped with 9mm clips. The superficial skin layer was closed using the FST system. Throughout the procedure and recovery, the animal was placed on a heating pad to maintain body temperature, and complications were monitored daily. No signs of pain or unusual painful behavior were observed during recovery.

[0165] [2.3. Mouse model of syngeneic endometriosis]

[0166] In this study, we used a second model of endometriosis, called the endometriosis syngeneic mouse model. This clinically relevant mouse model is generated by inoculating uterine horn fragments into the peritoneal cavity, as described in the publication cited in this paper

[21] . This is a minimally invasive model because the inoculated fragments are randomly distributed throughout the peritoneal cavity, where they implant and develop into lesions that mimic the natural occurrence of human endometriosis lesions

[21] . The model developed lesions and extensive chronic pelvic pain, both hallmarks of human endometriosis. [2.4. Syngeneic models of endometriosis (Endo) and Sham procedure]

[0167] Seven days after ovariectomy, endometriosis was induced in recipient mice by inoculating fragments of uterine horn tissue collected from donor mice into recipient mice. To collect donor tissue, the uterine horn was excised and clamped into a dissecting dish (Sigma-Aldrich, St. Louis, MO) containing 0.5 ml of ice-cold sterile phosphate-buffered saline (PBS) with penicillin (100 U / ml) and streptomycin (100 μg / ml). The uterine horn was longitudinally dissected and cut into small fragments. Recipient mice were then anesthetized with isoflurane and given a low dose (0.05 mg / kg) of the analgesic buprenorphine before induction. Under aseptic conditions, a small midline abdominal incision was made to expose the peritoneum, and half of the collected donor uterine horn tissue fragments (total volume ~0.2 ml of fragment-containing solution) was inoculated into the peritoneal cavity of one recipient mouse using a 1 ml pipette. For the Sham procedure, a similar procedure was performed, but the patient was inoculated with 0.2 ml of sterile phosphate-buffered saline (PBS) containing penicillin (100 U / ml) and streptomycin (100 μg / ml) (Sigma-Aldrich, St. Louis, MO). Subsequently, the abdominal muscles were sutured and clamped with 9 mm clips. The superficial skin layer was closed using the FST system. Throughout the procedure and recovery, the animal was placed on a heating pad to maintain body temperature, and complications were monitored daily. No signs of pain or unusual painful behavior were observed during recovery.

[0168] [3. Characteristics of the growth and development of endometriosis lesions]

[0169] After all behavioral assays were completed, endometriotic lesions growing from surgically transplanted uterine horn fragments were collected and measured to track the development of endometriotic lesions following treatment with control Ab or NP137. All tissues were collected at least 24 hours after the last treatment with control Ab or NP137 to minimize any acute effects of either treatment, and all tissues were collected at least 3 days after estradiol administration to minimize estrogen variability between time points.

[0170] 3.1. Collection and measurement of endometriotic lesions

[0171] Following necropsy, lesions in Endo (control Ab) and Endo (NP137) mice were located, dissected, measured, and weighed. Four endometriosis lesions were collected from each mouse (2 × uterine lesions and 2 × uterine horn lesions). Lesions damaged during dissection or unable to be cleanly separated from surrounding tissues were excluded from lesion growth analysis. Measurements were taken at the lesion's maximum point using calipers (Castroviejo calipers 8.5 cm / 3-1 / 4").

[0172] 3.2. Statistical analysis of lesion growth

[0173] Data are expressed as mean ± SEM, where N represents the number of animals per group and n represents the number of individual lesions per group. Data were plotted using Prism 9 software (GraphPad, San Diego, CA, USA). For two groups with unequal variances, the Mann-Whitney nonparametric test was used for statistical analysis. Differences were considered statistically significant at **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0174] [4. In vivo assessment of chronic pelvic pain by quantifying visceral motor response (VMR)]

[0175] Visceral motor response (VMR) is a nociceptive brainstem reflex consisting of abdominal muscle contractions in response to noxious distension of hollow organs such as the vagina and colon [4, 6, 7, 19, 25, 27, 16]. We have documented VMR for vaginal distension (VD) or colonic distension (CRD) as an objective measure of pain sensitivity of the vagina and colon in fully conscious animals [14, 3, 22, 24, 5, 2, 8]. Using this technique, we have previously shown that mice with endometriosis develop both colonic and vaginal hypersensitivity [9, 21]. To determine whether long-term treatment with NP137 reduces vaginal and colonic hypersensitivity developed in a mouse model of endometriosis, we used VMR to determine any differences between Sham mice (control Ab) and Endo mice (control Ab or NP137). All mice were studied at least 24 hours after their last treatment with either Ab or NP137 to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen variability between time points.

[0176] [4.1. Surgical implantation of electromyography (EMG) electrodes]

[0177] VMR is objectively assessed by recording the electrical activity (electromyography (EMG)) generated by abdominal muscle contractions in response to harmless and harmful vaginal or colorectal distension. To achieve this, three days prior to VMR, under isoflurane anesthesia, the exposed ends of two PTFE-coated stainless steel wires (Advent Research Materials Ltd, Oxford, UK) were sutured into the right external oblique abdominal muscle of Sham or Endo mice, passing subcutaneously and exiting at the base of the neck for future use. At the end of the procedure, the mice received prophylactic antibiotics (…). Administer 5 mg / kg subcutaneously and analgesics (buprenorphine, 0.5 mg / 10 kg subcutaneously), then return to their respective cages and allow them to recover for at least 3 days, as previously described [21, 9, 8].

[0178] 4.2. Assess the visceral motor response (VMR) to vaginal dilation (VD) or colorectal dilation (CRD)

[0179] On the day of VMR assessment, mice were temporarily anesthetized with inhaled isoflurane, and a saline enema (100 μL) was administered into the colon / rectum or vagina (50 μL) via catheter. A balloon was then inserted into the colon / rectum or vagina for VMR assessment, as previously described [21, 9, 6, 7]. In short, a lubricated balloon was gently inserted through the anus into the colon / rectum until it extended 0.25 cm beyond the anal margin (2.5 cm long latex balloon) for CRD, or gently passed through the vagina and inserted 1 mm proximal to the vaginal margin (3 mm long latex balloon) for VD. The balloon catheter was secured to the tail root with surgical tape and connected to a constant pressure device (Isobar 3, G&J Electronics, Willowdale, Canada) for graded and pressure-controlled balloon dilation. Mice were gently restrained in mouse restraints with a dorsal channel and allowed to recover from anesthesia for 10 minutes before beginning the dilation sequence. Pre-set and via a constant voltage, apply 20-40-50-60-70-80 mmHg (20-second duration) at 4-minute intervals for CRD, or 20-30-40-60-70-80 mmHg (30-second duration) at 3-minute intervals for VD. The EMG electrodes are forwarded to a data acquisition system and the signals are recorded (NL100AK headstage), amplified (NL104), filtered (NL 125 / 126, Neurolog, Digitimer Ltd, bandpass 50–5000 Hz), and digitized (CED 1401, Cambridge Electronic Design, Cambridge, UK) to a PC for offline analysis using Spike2 (Cambridge Electronic Design), as described above [6]. Mice are allowed to recover from their first expansion protocol (randomized to VD or CRD) for at least 3 hours before the next expansion protocol. After the final expansion, the mice were returned to their IVC cages and put back on the rack for final intraperitoneal treatment and tissue collection.

[0180] [4.3. Statistical analysis of VMR in response to VD and CRD]

[0181] Simulated EMG signals were rectified and integrated for VMR analysis. To quantify the size of the VMR at each expansion pressure, the AUC during expansion was corrected for baseline activity (area under the curve (AUC) before expansion). The AUC was quantified by calculating the AUC of the EMG signal at each expansion pressure. Furthermore, the sum of all AUCs at the full expansion pressure (total AUC) was analyzed as a population parameter for the VMR response of each individual mouse. VMR data are expressed as mean ± SEM, where N represents the number of animals. Analyses and graphs were created in GraphPad Prism software (version 9, San Diego, CA, USA). Statistical analysis of the VMR data for the AUC at each expansion pressure was performed using the generalized estimating equation (GEE), followed by a post-hoc minimum significance (LSD) test using SPSS 23.0 where appropriate. Statistical analysis of the total AUC data was performed using Prism9 software (GraphPad software, San Diego, CA, USA). Where appropriate, ordinary one-way ANOVA and Sidak's multiple comparison post-hoc test were used for groups with homogeneous variances; or Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0182] [5. In vivo assessment of induced skin sensitivity to heat and mechanical stimuli]

[0183] To determine whether long-term NP137 treatment reduced elevated induced pain-like behaviors developed in a mouse model of endometriosis, we assessed skin sensitivity using both the hot plate test (thermal sensitivity) and the electronic von Frey (EvF) test (mechanical sensitivity). To ensure consistency and minimize bias, all measurements were performed by the same investigator in a blinded manner. All mice were studied at least 24 hours after the last treatment with either the control Ab or NP137 to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen variability between time points.

[0184] 5.1. Assessing skin sensitivity to mechanical stimuli using the electronic von Frey (EvF) reflex.

[0185] The mechanical aberration of pain from mechanical stimulation was assessed by measuring withdrawal in response to normally painless stimuli using the electronic von Frey (EvF) test

[12] . We previously used this technique to demonstrate the development of mechanosensitivity in a mouse model of endometriosis [9]. Prior to the EvF test, mice were acclimatized to a shell made of transparent Plexiglas (observation chamber approximately 230 x 240 x 146 mm, BSBIOEVF, Panlab, Spain) and placed on an elevated wire mesh rack for 30 minutes daily for two days. On the day of the test, mice were transferred to the test chamber in their IVC and acclimatized for at least 15 minutes. Animals were placed alone in the test area (with a breathable cover on top) and left undisturbed for 15 minutes. The EvF unit (handheld force sensor, BSBIOEVF4s, Panlab, Spain) was fitted with a semi-flexible tip and zeroed. Once the animal was stationary, the force sensor was applied vertically from below to the animal's hind paw or lower abdomen. The force was gradually increased until a noticeable withdrawal response was observed. The maximum force (in grams) that caused the paw or abdomen to retract was recorded as the retraction threshold. Each area was stimulated 5 times, and the average value was recorded as the experimental retraction threshold for each mouse. At the end of the test, the mice were returned to their cages and placed back on their IVC racks.

[0186] Statistical Analysis

[0187] The average force (in grams) required to elicit a withdrawal response using EvF was recorded for analysis. Data are expressed as mean ± SEM, and N represents the number of animals. Statistical analysis was performed using Prism 9 software (GraphPad, San Diego, CA, USA), and, where appropriate, ordinary one-way ANOVA and Turkey's post-hoc test for homogeneous variances were used; or Kruskal-Wallis test and Dunn's multiple comparisons were used for heterogeneous variances. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0188] [5.2. Assessing skin sensitivity to harmful heat stimuli using hot plate delay time]

[0189] The hot plate test is a commonly used test for measuring acute heat injury perception by monitoring reflex behavior to heat stimuli. Using this technique, we have previously demonstrated the development of heat hypersensitivity in a mouse model of endometriosis [21, 9]. Before testing, mice were moved to the test chamber in their IVC and allowed to acclimatize for at least 15 minutes. Mice were then placed individually on a preheated hot plate (LE7406, Harvard Apparatus, Panlab, Spain) set at 54 ± 1 °C (proven to be a noxious stimulus) and surrounded by a transparent methacrylate protective shell. The time from placement on the hot plate to the appearance of the first signs of pain response (including licking, flicking, or jumping, whichever comes first) was recorded as their hot plate delay time. Mice were allowed to recover in their cages for 24 hours and the test was repeated. The average response delay time of the two tests was recorded as the final hot plate delay time. Mice were then returned to their cages and IVC racks.

[0190] Statistical Analysis

[0191] The mean response (in seconds) between two exposures to the hot plate was recorded for analysis. Data are expressed as mean ± SEM, and N represents the number of animals. Statistical analysis was performed using Prism 9 software (GraphPad, San Diego, CA, USA), and the Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0192] 6. In vivo assessment of spontaneous behavior

[0193] To determine whether long-term NP137 treatment reduced enhanced spontaneous pain-like behavior developed in a mouse model of endometriosis, we used voiding pattern analysis and overnight nesting analysis to identify any differences between Sham mice (control Ab) and Endo mice (control Ab or NP137). All mice were evaluated at least 24 hours after the last treatment with either control Ab or NP137 to minimize any acute effects of either treatment, and at least 3 days after estradiol administration to minimize estrogen variability between time points. To ensure consistency and reduce bias, all measurements were performed blinded by the same investigator. At the end of each test protocol, mice were returned to their cages and placed back on their IVC racks for subsequent measurements.

[0194] 6.1. In vivo assessment of bladder dysfunction through voiding pattern analysis

[0195] Voiding pattern analysis is a voiding assessment tool that provides information on changes in spontaneous behavioral patterns associated with urinary tract pathologies, including overactive bladder (OAB) and interstitial cystitis / bladder pain syndrome (IC / PBS) [16, 17]. Voiding pattern analysis was performed as previously described to assess changes in bladder function following the development of endometriosis in mice [9, 21].

[0196] Urination pattern analysis was performed on all mice 8 weeks after endometriosis or Sham induction surgery and 4 weeks after control (Ab or NP137) treatment. For this purpose, all bedding was removed from each individual Endo and Sham mouse cage, and filter paper was placed at the bottom of each cage. Mice remained in the cages with filter paper for 3 hours between 9:00 AM and 12:00 PM, with free access to food and water. During this period, spontaneous urination patterns were generated on the filter paper. The filter paper was collected and preserved for imaging, the bedding was returned to the IVC (intrauterine cavity), and the mice were returned to the IVC rack.

[0197] Urine samples collected from filter paper were imaged using a UV-transmitted illuminator (Bio-Rad, California, USA) and digitized into binary images using ImageJ software (NIH, ImageJ, 2.0.0). The number and size of urine stains were determined using preset thresholds in ImageJ software, and for the purposes of this study, the number of small stains (100–1,000 pixels), medium stains (1,001–100,000 pixels), and large stains (>100,000 pixels) were quantified.

[0198] Statistical Analysis

[0199] Data are expressed as mean ± SEM, where N represents the number of animals. Data were plotted using Prism 9 software (GraphPad, San Diego, CA, USA). Where appropriate, ordinary one-way ANOVA and Turkey's post-hoc test for multiple comparisons were used for groups with homogeneous variances; or Kruskal-Wallis test and Dunn's multiple comparisons were used for groups with unequal variances. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0200] [6.2. Assessing the overall health status of animals through spontaneous overnight nesting analysis]

[0201] Spontaneous nesting analysis is a sensitive assessment tool that provides information on changes in spontaneous behavioral patterns related to overall health in mice

[13] . In this study, we used a predefined 5-point scale for overnight nesting analysis to assess the effects of long-term NP137 doses on overall animal health, as previously described

[11] .

[0202] Nesting patterns were analyzed in all mice 8 weeks after endometriosis or Sham induction surgery and 4 weeks after control Ab or NP137 treatment. For this purpose, all environmental enrichment items (including original nesting material, cardboard tunnels and paper towels) were removed from each individual Endo and Sham mouse cage, leaving only the original bedding material at the bottom of each cage. Pre-weighed nesting material (5 cm square cotton pads - about 3 g) was placed in each cage, and the mice were placed back on the IVC rack and allowed to nest undisturbed overnight (4 p.m. to 9 a.m.). The next morning, individual nests were evaluated blinded and scored according to a pre-defined 5-point nesting rating scale (range 1–5), where 1 indicates that the nesting material was not touched and 5 indicates that the nesting material was completely torn and had high and fluffy walls, as described above

[11] . The environmental enrichment items were returned to the cages, and the mice were placed back on their IVC racks.

[0203] Statistical Analysis

[0204] Data are expressed as mean ± SEM, where N represents the number of animals. Data were plotted using Prism 9 software (GraphPad, San Diego, CA, USA), and analyzed using the Kruskal-Wallis test and Dunn's multiple comparisons. Differences were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0205] 【7. Results and Conclusions】

[0206] [7.1. Mice with endometriosis develop cystic endometriosis lesions]

[0207] The mouse model of endometriosis used in this study was a modification of a previously established model of endometriosis induced by surgical transplantation [9]. In this model, uterine horn fragments were transplanted to each side of the uterine fornix and the end of the uterine horn, as described in the Methods section (and also as...). Figure 2 A1). Control mice (Sham mice) were produced after the same surgical procedure but without uterine horn tissue ( Figure 2 A1). As previously mentioned [9], the transplanted fragments developed into endometriotic lesions 10 weeks after transplantation (see also...). Figure 2 A1).

[0208] 7.2. NP137 treatment reduces the progression of endometriosis lesions.

[0209] To investigate whether NP137 treatment could limit lesion development and provide analgesia in mice with endometriosis, we examined the growth of endometrial lesions. The efficacy of NP137 treatment was quantified by comparing the extent of endometriosis development in Endo mice treated with NP137 with that observed in Endo mice treated with the control antibody NP001 (control Ab). Figure 2 A1). As previously mentioned, Sham mice did not develop endometrial lesions at the surgical suture site. Figure 2 A1).

[0210] Examination of the female reproductive tracts of mice with endometriosis showed that long-term NP137 treatment reduced the size of the developing lesions. Figure 2 A1-2). The reduction in lesion size... Figure 2 Visually shown on A2, this figure depicts endometrial lesions collected from four Endo mice treated with NP001 (control Ab). Figure 2 A2 (left image) and endometrial lesions collected from three Endo mice treated with NP137 ( Figure 2 A2 (right figure). Quantitative analysis of the collected lesions showed that NP137 treatment significantly reduced the weight and size of these endometriosis lesions. Figure 2 (B~C). Interestingly, the NP137-induced lesion size reduction is specific to lesions developing around the uterus, rather than lesions growing at the distal end of the uterine horn. Figure 2 A2 and 2B-C). Notably, a single treatment group (four additional Endo mice treated with NP001 and five Endo mice treated with NP137) confirmed the preliminary findings, indicating that NP137 significantly reduced the lesion size in mice with Endo. Figure 11 A1-2). Furthermore, data from these additional mice will be included in the overall data (A1-2). Figure 11 B~C), eliminating the regional differences observed in the first group of treatments ( Figure 2 A2 and 2B-C). Therefore, NP137 can effectively reduce the development of endometriosis lesions, regardless of their location. Figure 11 B2~3 and 11C2~3).

[0211] Interestingly, we also tested the effect of NP137 on reducing lesions that develop in different mouse models of endometriosis, known as endometriosis syngeneic mouse models. Figure 10This is a minimally invasive model, similar to the natural occurrence of endometriosis lesions in humans, because the inoculated fragments are randomly distributed throughout the peritoneal cavity, where they implant and develop into lesions. Notably, NP137 treatment tends to reduce the size and number of lesions developing within the peritoneal cavity in syngeneic mouse models of endometriosis. Figure 10 (A~C). This indicates that NP137 can not only effectively reduce the growth of lesions after their formation, but also potentially inhibit the formation of lesions themselves.

[0212] [7.3. NP137 treatment reduces vaginal hyperalgesia developed in endometriosis]

[0213] Visceral pain is measured in vivo by recording the visceral motor pain response (VMR) induced by vaginal dilation (VD). VMR is the gold standard for measuring pain induced by hollow organs such as the vagina [3, 26] and the colon (based on our previous studies [6, 16]). The VMR response is quantified by recording electromyographic (EMG) activity from electrodes surgically implanted in the abdominal muscles of mice, as previously described [8].

[0214] In Sham mice, vaginal dilation increases the VMR response, and the degree of VMR is related to the applied pressure. Figure 3 A). We found that mice with fully developed endometriosis responded to 20 mmHg and 40–70 mmHg of vitamin D (VD). Figure 3 A) and for all expansion combinations ( Figure 3 B) showed a significantly increased VMR response; indicating the development of vaginal hyperalgesia in endometriosis mice. Notably, treatment with NP137 significantly reduced the VMR response in Endo mice compared with those treated with control Ab, particularly at lower (<40 mmHg) distending pressures. Figure 3 C). Furthermore, the overall effect of NP137 is to normalize the VMR response to the VMR response observed in Sham control mice (C). Figure 3 D).

[0215] Overall, these results indicate that treatment with the monoclonal antibody NP137 is an effective method for relieving vaginal hyperalgesia associated with endometriosis.

[0216] 7.4. NP137 treatment reduces colorectal hyperalgesia that develops in endometriosis.

[0217] Women with endometriosis are often diagnosed with other chronic visceral complications, such as irritable bowel syndrome (IBS)

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

[0218] We found that, compared with Sham mice treated with the control Ab, Endo mice treated with the control Ab showed better control of all expansions in the tested range of 20–80 mmHg. Figure 4 A) and for all expansion combinations ( Figure 4 B) Exhibited a significant hypersensitivity response to CRD; indicating the development of colorectal hyperalgesia in endometriosis mice. Similar to what was observed with VD, treatment with NP137 significantly reduced the VMR response in Endo mice compared to those treated with control Ab, particularly at lower (<40 mmHg) distending pressures. Figure 4 C). Furthermore, the overall effect of NP137 is to normalize the VMR response to the VMR response observed in Sham control mice (C). Figure 4 D).

[0219] Importantly, these findings are clinically relevant because women with endometriosis often experience severe CPP involving the colon and vagina [20, 29]. Overall, these results suggest that treatment with a monoclonal anti-axonal guidance factor antibody (NP137) is an effective approach to relieving endometriosis-associated colorectal hyperalgesia.

[0220] 7.5. NP137 treatment normalizes bladder function changes that develop in endometriosis.

[0221] Women with endometriosis often suffer from visceral complications, including overactive bladder (OAB) and interstitial cystitis / bladder pain syndrome (IC-PBS). In this study, we aimed to determine whether our mouse model of endometriosis developed bladder function alterations, and importantly, whether NP137 treatment could normalize function. To investigate this, we examined and compared spontaneous bladder voiding patterns in Sham and Endo mice treated with control Ab with those in Endo mice treated with NP137, as an indicator of bladder function alterations.

[0222] We found that mice with fully developed endometriosis exhibited altered bladder emptying patterns compared to Sham mice, as evidenced by an increase in the total number of urinary spots. Figure 5 A). This disordered, scattered urination pattern is characterized by an increased number of small urinary spots (A). Figure 5B), indicating OAB / IC-PBS symptoms of urinary urgency and frequency

[17] .

[0223] Interestingly, NP137 treatment normalized bladder dysfunction developed in mice with endometriosis, and a reduction in the total number of urinary spots was observed in Endo mice treated with NP137. Figure 5 A) proves this point, especially with small-sized urine stains ( Figure 5 B). Furthermore, the overall effect of NP137 was to normalize the urination pattern to that observed in Sham control mice. Figure 5 (A~B).

[0224] 7.6. NP137 treatment normalizes the increased skin sensitivity to heat and mechanical stimuli that develops in endometriosis.

[0225] In addition to comorbidities related to internal organs, women with endometriosis also experience altered skin sensation [18, 28]. Here, we determined whether our mouse model of endometriosis exhibited altered skin sensitivity and whether NP137 treatment could correct it. To determine whether the development of endometriosis affects skin sensitivity induced by thermal and mechanical stimuli, mice underwent hot plate and electronic von Frey (EvF) tests.

[0226] We found that mice with fully developed endometriosis treated with control Ab showed enhanced sensitivity to mechanical stimulation of their hind paws, indicating a significantly reduced force required to elicit a response compared to their Sham counterparts. Figure 6 A). The force required to elicit a response is called the mechanical threshold, and a decrease in this threshold indicates increased sensitivity to mechanical stimuli. We also found that mice treated with Endo control Ab showed increased sensitivity to thermal stimuli, indicating a significantly reduced response time to the hot plate compared to their Sham counterparts. Figure 6 B). These results demonstrate that, compared to Sham mice, our mouse model of endometriosis did indeed experience enhanced sensitivity to mechanical and thermal stimuli of the skin.

[0227] Interestingly, we found that treatment with NP137 effectively reversed the increased sensitivity to mechanical and thermal stimuli observed in Endo mice treated with control Ab. Figure 6 (A-B). Importantly, the observed reduction levels were similar to those observed in Sham mice treated with control Ab (A-B). Figure 6(A-B). Notably, a separate treatment group (an additional 6 Endo mice treated with NP001 and 6 Endo mice treated with NP137) confirmed the preliminary findings, indicating that NP137 significantly reduced skin mechanical and thermal hypersensitivity in mice with Endo disease. Figure 12 (A~B).

[0228] Furthermore, we found that Endo mice (a syngeneic mouse model) induced by inoculating uterine horn fragments into the peritoneal cavity exhibited discretely enhanced sensitivity to thermal stimulation, indicating a reduced discrete delay in response time to the hot plate compared to the Sham corresponding group. Figure 8 Interestingly, we found that treatment with NP137 tended to reverse this effect observed in Endo mice treated with control Ab. Figure 8 ).

[0229] [7.7. NP137 improves signs of overall health decline in endometriosis]

[0230] Finally, we tested whether the development of endometriosis altered spontaneous behavior in a way that indicated a deterioration in the animal’s overall health, and whether NP137 treatment helped maintain the animal’s overall health. Spontaneous nesting analysis is a sensitive assessment tool that provides information on changes in spontaneous behavioral patterns related to the overall health of mice

[13] .

[0231] In this study, we analyzed the overnight nesting behavior of Sham and Endo mice treated with control Ab and found that mice with fully developed endometriosis had reduced nesting ability during this period. Figure 7 The reduced nesting activity indicates a lack of nocturnal activity, suggesting that the development of endometriosis reduces the animals' health. We found that treatment with NP137 could partially reverse nesting behavior in mice with endometriosis. Figure 7 ), the nesting behavior of Endo mice treated with NP137 was similar to that shown in Sham mice. Figure 7 Notably, a separate treatment group (an additional 6 Endo mice treated with NP001 and 5 Endo mice treated with NP137) confirmed the preliminary findings, indicating that NP137 significantly improved the overall health of the Endo mice. Figure 9 Incorporating data from these additional mice into the overall data showed that NP137 treatment completely reversed the overnight nesting deficits observed in mice with Endo disease. The nesting behavior patterns exhibited by Endo mice treated with NP137 were similar to those observed by Sham mice treated with NP001. Figure 9This suggests a beneficial effect on reducing anxiety-like behaviors associated with endometriosis.

[0232] 【7.8. Conclusion】

[0233] Overall, the results of this preclinical study demonstrate that long-term treatment with NP137 (a clinically approved mAb against axonal guidance factor-1) is effective in reducing the size of endometriotic lesions developed in a clinically relevant mouse model of endometriosis. Furthermore, NP137 treatment reversed alterations in pain sensitivity in multiple peripheral organs, thereby reducing chronic pelvic pain experienced by mice with fully developed endometriosis. These results strongly suggest that NP137 is a viable therapeutic strategy for women with endometriosis to reduce or eliminate endometriotic lesions and alleviate chronic pelvic pain.

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Claims

1. Anti-axon guide factor-1 antibody or its antigen-binding fragment, for use in the treatment of endometriosis, said treatment comprising reducing endometriotic lesions, particularly endometriotic lesions in the uterus and / or uterine horns.

2. The antibody or its antigen-binding fragment for the purpose according to claim 1, wherein, The treatment also includes relief of chronic pelvic pain.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The treatment also includes relief of vaginal hyperalgesia.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein, The treatment also includes: • Reduced colorectal hyperalgesia developed in endometriosis; • Improvement of bladder dysfunction developed in endometriosis; • Decreased skin sensitivity to increased thermal and / or mechanical stimuli, which develops in endometriosis; • Improvement in overall health due to endometriosis changes.

5. An anti-axon guidance factor-1 antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein, The antibody or antigen-binding fragment specifically binds to the polypeptide of sequence SEQ ID NO:

33.

6. An anti-axon guidance factor-1 antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein, The antibody is a monoclonal antibody or its antigen-binding fragment, wherein the antibody or fragment comprises: A variable structural domain VH, which contains: H-CDR1, which has the sequence shown in SEQ ID NO: 5; H-CDR2, which has the sequence shown in SEQ ID NO: 6; H-CDR3, which has the sequence shown in SEQ ID NO: 7; A variable domain VL, which contains: • L-CDR1, which has the sequence shown in SEQ ID NO: 8; L-CDR2, which has the sequence YAS; • L-CDR3, which has the sequence shown in SEQ ID NO: 9; or A variable structural domain VH, which contains: H-CDR1, which has the sequence shown in SEQ ID NO: 28; • H-CDR2, which has the sequence shown in SEQ ID NO: 29; • H-CDR3, which has the sequence shown in SEQ ID NO: 30; A variable domain VL, which contains: • L-CDR1, which has the sequence shown in SEQ ID NO: 31; • L-CDR2, which has the sequence shown in SEQ ID NO: 32; ·L-CDR3, which has the sequence shown in SEQ ID NO:

9.

7. An anti-axon guidance factor-1 antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein, The antibody is a monoclonal antibody or its antigen-binding fragment, and The antibody or fragment thereof comprises VH and VL sequence pairs 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 VH and VL sequence pairs SEQ ID NO: 22 and 16.

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

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

16.

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

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