Migraine Therapy

Abstract

The present disclosure generally provides compositions and methods useful for treating migraine. More specifically, the methods and compositions can be used to treat, alleviate, or prevent migraine in subjects who are poor responders to anti-5CGRP therapy (aCGRP-IR) or who do not respond to anti-CGRP (calcitonin gene-related peptide) therapy or to inhibitors of calcitonin gene-related peptide (CGRP).

Description

[Technical Field]

[0001] Provided herein are methods for treating migraine in migraine patients who are either inadequate responders or non-responsive or intolerant to anti-CGRP therapy or inhibitors of CGRP, comprising administering to the patient an anti-PAR2 antagonist antibody. [Background technology]

[0002] Migraine is a common neurological disorder characterized primarily by headache in the absence of trauma, which can be severe and is often localized to one side of the head. Migraines are generally recurrent and occur frequently in some individuals. Recurrent migraines can be severely debilitating for sufferers, and according to the UK National Health Service, migraines affect approximately 20% of adult women (and more than 5% of adult men), resulting in a substantial economic and social burden.

[0003] The mechanisms underlying migraine headaches are not fully understood, but they are complex and likely involve both environmental and genetic factors. It is known that migraine pain is ultimately caused by activation of nociceptors in the brain meninges, which is thought to be triggered by degranulation of immune mast cells in the dura mater. Mast cell degranulation leads to the release of mediators, including serine proteases such as tryptase and trypsin, which induce the release of neuropeptides, including the neuropeptide calcitonin gene-related peptide (CGRP), from nerve terminals (Aich et al., International Journal of Molecular Sciences 16:29069-29092, 2015). CGRP may indirectly mediate nociceptor activation (Kopruszinski et al., Cephalalgia 40(14):1535-1550, 2020).

[0004] CGRP release is induced by serine proteases via the G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2). The PAR family is known to contain four members (PAR1-PAR4), and their activation has been found to be associated with inflammation and nociception (Gieseler et al., Cell Communication and Signaling 11:86, 2013). PARs, including PAR2, are activated by proteolytic cleavage of the extracellular domain of the PAR, which acts to expose an N-terminal "tethered ligand," i.e., a segment of the extracellular N-terminus of the PAR that binds to the receptor's activation sequence after cleavage. Binding of the tethered ligand to the PAR's activation sequence results in receptor activation. PAR2 is activated by serine proteases, including trypsin and tryptase (Gieseler et al., supra).

[0005] PAR2 has previously been proposed as a target for therapy for migraine and other types of pain, and WO2018 / 167322 discloses several PAR2-specific antibodies, including MEDI0618, that antagonize the receptor and may be therapeutically useful.

[0006] Currently, when standard analgesics such as paracetamol or ibuprofen are ineffective in treating a patient's migraine, first-line treatments include triptans, beta-blockers (e.g., propranolol, topiramate (a carbonic anhydrase inhibitor), and candesartan (an angiotensin receptor blocker). For patients in whom such first-line treatments are ineffective or inappropriate, second-line treatments, such as calcium channel blockers, tricyclic antidepressants, or valproic acid (or its salts), may be prescribed.

[0007] For patients in whom second-line treatment is ineffective or inappropriate, newer third-line treatments have recently become available, and currently consist primarily of drugs that target the CGRP signaling axis. These third-line drugs include gepants (CGRP receptor antagonists) and antibodies against CGRP and the CGRP receptor. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2018 / 167322 [Non-patent literature]

[0009] [Non-Patent Document 1] Aich et al., International Journal of Molecular Sciences 16:29069-29092, 2015 [Non-patent document 2] Kopruszinski et al., Cephalalgia 40(14):1535-1550, 2020 [Non-patent document 3] Gieseler et al., Cell Communication and Signalling 11:86, 2013 Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have found that, although PAR2 acts within the CGRP signaling pathway, PAR2 inhibition is effective in preventing both CGRP-dependent and CGRP-independent migraine pain (the pathway by which CGRP-independent migraine pain occurs has not yet been elucidated). [Means for solving the problem]

[0011] (Summary of the Invention) In a first aspect, provided herein is a method for treating, alleviating, or preventing migraine in a subject, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of protease-activated receptor 2 (PAR2); The method is one in which the subject is an inadequate responder to anti-CGRP therapy (aCGRP-IR) or the subject suffers from migraine that is unresponsive to anti-CGRP therapy or an inhibitor of calcitonin gene-related peptide (CGRP).

[0012] In a second aspect, provided herein is a method for treating, alleviating, or preventing migraine in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to and inhibits activity of protease-activated receptor 2 (PAR2); The method is one in which the subject is intolerant to anti-CGRP (calcitonin gene-related peptide) therapy or an inhibitor of calcitonin gene-related peptide (CGRP).

[0013] In a third aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject suffering from migraine that is unresponsive to anti-CGRP therapy or an inhibitor of calcitonin gene-related peptide (CGRP).

[0014] In a fourth aspect, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject suffering from migraine and who is intolerant to anti-CGRP (CGRP) therapy or an inhibitor of calcitonin gene-related peptide (CGRP).

[0015] In a fifth aspect, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 in the manufacture of a medicament for the treatment of migraine in a subject suffering from migraine that is unresponsive to therapy with anti-CGRP therapy.

[0016] In a sixth aspect, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 in the manufacture of a medicament for the treatment of migraine in a subject suffering from migraine and who is intolerant to inhibitors of calcitonin gene-related peptide (CGRP). [Brief explanation of the drawings]

[0017] [Figure 1A] Figure 1 shows the effect of olcegepant (1 mg / kg, i.p.) on migraine-like pain behavior induced by epidural administration of inflammatory mediators (IM) (A) or CGRP (B). Pain was measured by the tactile frequency of response (%) to von Frey hairs applied to the periorbital area. Olcegepant was injected 30 min before either IM or CGRP. [Figure 1B] Figure 1 shows the effect of olcegepant (1 mg / kg, i.p.) on migraine-like pain behavior induced by epidural administration of inflammatory mediators (IM) (A) or CGRP (B). Pain was measured by the tactile frequency of response (%) to von Frey hairs applied to the periorbital area. Olcegepant was injected 30 min before either IM or CGRP. [Figure 2A-F]Figure 1 shows the effect of MEDI0618 on migraine pain-like behavior induced by epidural administration of inflammatory mediators (IM) in female mice. Data represent the frequency of responses to tactile stimulation of von Frey hairs applied to either the periorbital or hind paw region. Antibody (mAb, 50 mg / kg) was injected subcutaneously at baseline (BL1). Inflammatory mediators were injected epidurally after baseline 2 (BL2). Statistical differences from control (BL2) are indicated by **(p<0.01) ***(p<0.001). [Figure 3] We demonstrate that PAR2 is functionally expressed in a distinct population of TG neurons that do not necessarily co-express CGRP receptors. Single-cell calcium imaging from adolescent mouse trigeminal ganglion neurons revealed increases in cytosolic calcium in response to a PAR2 agonist (2-furoyl-LIGRLO-amide, 10 μM) in 24% of responding neurons across four biological n. Only 14% of these PAR2 agonist-activated neurons were co-activated by CGRP (1 μM). The left panel shows individual cell data, with arrows indicating the time point at which the agonist was applied. "Neuron Stim" indicates the addition of high potassium buffer (20 mM) to stimulate all neurons. The right panel shows average data from two independent cultures demonstrating the mean fold change from baseline in cellular calcium signals. [Figure 4A] These results show that MEDI0618 demonstrates superior potency and efficacy compared to PAR650097 (Kopruszinski et al., Cephalagia) in blocking PAR2-mediated calcium flux in an endogenous hPAR2 calcium imaging assay. Human dural fibroblasts and dural microvascular endothelial cells were loaded with the calcium indicator dye Fluo-8 and pretreated with the indicated concentrations of mAb. Peak fluorescence was measured in response to the addition of matriptase (30 nM). Isotype control antibody = NIP 229 hIgG1. [Figure 4B]These results show that MEDI0618 demonstrates superior potency and efficacy compared to PAR650097 (Kopruszinski et al., Cephalagia) in blocking PAR2-mediated calcium flux in an endogenous hPAR2 calcium imaging assay. Human dural fibroblasts and dural microvascular endothelial cells were loaded with the calcium indicator dye Fluo-8 and pretreated with the indicated concentrations of mAb. Peak fluorescence was measured in response to the addition of matriptase (30 nM). Isotype control antibody = NIP 229 hIgG1. [Figure 5] The improved pharmacokinetic properties of MEDI0618 compared to related antibodies are shown. Due to target-mediated clearance, the anti-PAR2 antibody P67 (PAR0067, MEDI2344) exhibited nonlinear pharmacokinetics at a dose of 1 mg / kg (iv) in rats, with a significantly higher systemic clearance rate (164 mL / day / kg) than expected for a typical monoclonal antibody. Histidine mutagenesis in the VHCDR2, VHCDR3, and VLCDR3 sequences of PAR0067 resulted in mAb clones H48-H139. Data show serum concentrations of mAb after iv injection of 1 mg / kg in rats. H129 = MEDI0618. DETAILED DESCRIPTION OF THE INVENTION

[0018] Presented herein are methods for treating, alleviating, or preventing migraine in a subject. "Treating, alleviating, or preventing migraine" may alternatively be referred to herein as "migraine therapy" or "therapy for migraine." These terms are used interchangeably herein. As noted above, the method includes administering to the subject an antibody, or an antigen-binding fragment thereof, that binds to and inhibits the activity of PAR2. "Subject," when referred to herein, may alternatively be referred to as "patient."

[0019] As used herein, "preventing" a migraine means that the method prevents the onset of a migraine after administration of an antibody (or fragment thereof) to a subject. Preventing a migraine includes completely preventing a migraine in a subject for a certain period of time after administration of the antibody or fragment thereof, i.e., administering the antibody to a subject can completely prevent the onset of a migraine in a subject for, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. If the therapy completely prevents the onset of a migraine in a subject for a certain period of time after administration of the antibody, the subject may not experience a migraine or migraine symptoms (e.g., pain or aura) during that period.

[0020] Preventing migraines also includes reducing the frequency of migraines suffered by a patient. The therapies provided herein can reduce the frequency of migraines or the number of migraine days suffered by a patient per month by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over a certain period of time after administration of an antibody or fragment thereof (compared to the frequency of migraines suffered by the patient before receiving the therapy). The reduction in migraine frequency in a subject can last, for example, for at least 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks, or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. For example, a patient suffering from migraines whose frequency is reduced after undergoing a therapy described herein may experience migraines about or less than once per week, about or less than once every two weeks, about or less than once every three weeks, about or less than once every four weeks, about or less than once every five weeks, or about or less than once every six weeks, or about or less than once per month, about or less than once every two months, about or less than once every three months, about or less than once every four months, about or less than once every five months, or about or less than once every six months.

[0021] "Treating" a migraine headache means that the methods provided herein are administered to a subject (i.e., an antibody or fragment thereof is administered to the subject) when the subject is experiencing migraine symptoms, thereby causing an improvement (i.e., a reduction or termination) of the symptoms. In this case, the antibody or fragment thereof may be administered to the subject at any time during the migraine episode, particularly at or around the time of symptom onset, but may also be administered later in the course of the migraine headache, thereby reducing the severity or completely eliminating the migraine symptoms. Thus, migraine treatment encompasses both complete and partial relief of ongoing migraine symptoms.

[0022] When partial relief of symptoms is achieved, the therapy achieves at least, e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the severity of the symptoms, as assessed by the subject or their physician. Any migraine symptom, e.g., headache, can be alleviated by the therapies provided herein.

[0023] The antibody or fragment thereof may be administered to a subject at the first sign of migraine symptoms in the subject to prevent the onset of a migraine, which can be considered both treating and preventing migraine.

[0024] By "alleviating" migraine headaches, it is meant that the therapies provided herein cause a partial, but not complete, reduction in the severity of migraine symptoms in a subject. Migraine relief can be achieved by administering an antibody or fragment thereof to a subject when the subject is suffering from a migraine (as described above in the Treatment section). In this case, migraine relief is achieved if a partial reduction in the severity of migraine symptoms results from the therapy. Such a partial reduction can be as described above, i.e., at least, e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in symptom severity, as assessed by the subject or their physician.

[0025] Migraine relief can also be assessed by the effect of the therapy on the subject's migraine-related disability and / or quality of life. Migraine-related disability is disability caused by migraine. A subject suffers from migraine-related disability if migraine prevents the subject from performing daily activities that would otherwise be normal for a person of the subject's age. For example, migraine-related disability may result in the inability to work or at least require the subject to reduce their working hours. Migraine-related disability may result in the inability to study or receive education or training. Migraine-related disability may result in the inability to perform household chores or care for oneself or children, or the inability to socialize or pursue hobbies. Such disability may severely impact the subject's quality of life and / or economic productivity. Migraine relief can reduce or even end the subject's migraine-related disability, such that the subject's migraine condition is no longer disabling, even if some migraine symptoms may occasionally appear and persist. For example, a subject may be able to return to work or work increased hours. A subject may be able to resume a hobby, exercise or play sports, perform household chores, care for children or other dependents, or generally have an improved ability to perform normal daily activities. A subject's quality of life may improve as a result, and may be measured qualitatively or quantitatively by a score-based rating or the like.

[0026] A reduction in the duration of migraine symptoms can also be considered migraine relief. For example, the duration of migraine symptoms can be reduced by the therapies provided herein, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, e.g., by at least 2, 4, 6, 8, 10, 12, 18, 24, 36, or 48 hours or more.

[0027] Similarly, if an antibody or fragment thereof is administered as a prophylactic therapy and does not completely prevent a subject from experiencing migraines, but reduces the frequency with which the subject experiences migraines and / or the severity or duration of the migraines they do experience, this can be considered migraine relief. The reduction in migraine frequency, severity, or duration achieved by the therapies provided herein can be of any magnitude, as described above. As will be apparent, there is overlap between "treatment," "alleviation," and "prevention" of migraines as defined herein, and thus the therapies provided herein may treat, alleviate, and / or prevent migraines in a subject.

[0028] subject The subject to be administered the therapy provided herein can be any subject who needs such therapy.In particular, the subject is the subject who suffers from migraine, particularly the subject who suffers from frequent and / or debilitating migraine (for example, particularly the migraine that lasts for a long time).For example, the subject can suffer from migraine at least about once a month, once every 3 weeks, once every 2 weeks or once a week, or about 2, 3, 4 or more times a week. In particular, the subject may be affected by migraine symptoms for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days per month (i.e., the subject may have at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 migraine days per month), for example, the subject may experience migraine headaches for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days per month. As noted above, the antibody or fragment thereof may be administered to the subject as a preventative therapy or as active therapy for ongoing migraine headaches.

[0029] Generally, the subject is a human, but may also be a non-human animal determined by a veterinarian to be suffering from migraines. For example, the subject may be a domestic animal, particularly a companion animal, such as a dog, cat, horse, or similar animal.

[0030] As noted above, in some embodiments of the methods provided herein, the subject suffers from migraine that is unresponsive to therapy with an inhibitor of calcitonin gene-related peptide (CGRP). In other embodiments of the methods provided herein, the subject is intolerant to inhibitors of CGRP.

[0031] Inhibitors of CGRP may be referred to as CGRP inhibitors, and the terms "CGRP inhibitor" and "inhibitor of CGRP" are used interchangeably herein. A CGRP inhibitor, as defined herein, is an agent that blocks the activity of CGRP, in particular, prevents CGRP from activating the CGRP receptor. CGRP inhibitors are further described below.

[0032] The therapeutic use of CGRP inhibitors may be referred to as anti-CGRP (aCGRP) therapy. aCGRP therapy includes antagonists to CGRP receptors and ligands, including both mAbs and non-peptide small molecules (gepants) used for acute and prophylactic treatment. The terms "anti-CGRP (aCGRP) therapy," "CGRP inhibitor," and "inhibitor of CGRP" may be used interchangeably herein.

[0033] Thus, the subject may suffer from migraine that is unresponsive to therapy with a CGRP inhibitor. Such subjects generally suffer from frequent and / or recurrent migraines and have received therapy with a CGRP inhibitor. Thus, in this embodiment, the subject has received at least one previous treatment for migraine, i.e., a CGRP inhibitor. As discussed further below, the subject may have received multiple previous migraine treatments before receiving therapy according to the methods provided herein. Such a subject may have received a single dose of a CGRP inhibitor, but more commonly, has received multiple doses of a CGRP inhibitor. The subject may have tried (i.e., been prescribed or administered) multiple (i.e., at least two) different CGRP inhibitors. The subject may also have tried multiple different dosage regimens of one or more CGRP inhibitors. For example, the subject may have tried multiple different dosage levels of one or more CGRP inhibitors, multiple different dosage frequencies, different routes of administration of one or more CGRP inhibitors, etc.

[0034] If a subject does not respond (or is non-responsive) to therapy with a CGRP inhibitor, it means that the CGRP inhibitor has not been found to effectively treat, alleviate, or prevent migraine headaches in that subject.

[0035] In some cases, a subject suffering from migraine that does not respond to CGRP inhibitor therapy may find that their condition does not improve during or after CGRP inhibitor therapy, i.e., the severity, duration, or frequency of their migraine headaches does not decrease in response to CGRP inhibitor therapy. That is, the subject may be a subject for whom CGRP inhibitor therapy is completely ineffective in treating migraine headaches. In this case, the subject's condition may not improve at all, i.e., the severity, duration, and frequency of the subject's migraine headaches may not change during and after CGRP inhibitor therapy compared to before receiving CGRP inhibitor therapy. If the subject is taking or receiving one or more additional therapies for their migraine headaches simultaneously with a CGRP inhibitor (e.g., analgesics such as paracetamol or ibuprofen, or other migraine treatments discussed below), the CGRP inhibitor will not provide any additional benefit beyond any benefit achieved by the other drugs. In some cases, the subject's condition may even worsen in response to CGRP inhibitor therapy, for example, the severity, duration, and / or frequency of the subject's migraine headaches may worsen.

[0036] Alternatively, the subject may experience only a slight or partial improvement in their condition in response to CGRP inhibitor therapy, such as a slight or partial reduction in the severity, duration, and / or frequency of migraines. For example, in response to CGRP inhibitor therapy, the subject may experience an improvement (i.e., reduction) of less than 30% (e.g., less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%) in the number of monthly migraine days (in the case of episodic migraine) or the frequency of monthly headache days (in the case of chronic migraine), and / or may remain adversely affected by the severity, duration, and / or frequency of migraines for 2, 3, 4, or 5 days or more per month. If the subject is taking or receiving one or more additional therapies for migraines simultaneously with a CGRP inhibitor, the slight improvement will exceed any benefits provided by the additional therapies. Such a subject may be considered by a physician to have responded inadequately to CGRP inhibitor therapy, e.g., the subject may not have responded as well to CGRP therapy as the physician could have predicted or hoped for. The subject may be considered by a physician to be in need of an alternative therapy to CGRP inhibitor therapy to treat, alleviate, or prevent migraine headaches.

[0037] For example, a subject may find that their condition has improved slightly as a result of CGRP inhibitor therapy, but not to the extent that their quality of life is improved or that they are able to carry out normal daily activities. For example, a subject may find that despite receiving CGRP inhibitor therapy, they are still unable to work, increase their work hours, do housework, or go outside or socialize, etc. (depending on their initial condition).

[0038] In a further alternative, the subject's migraine may have become unresponsive to therapy with a CGRP inhibitor. In this case, therapy with a CGRP inhibitor may initially be successful in treating, alleviating, or preventing the subject's migraine (as described above), but after an initial response, the subject's migraine may have returned or worsened, e.g., returned to its original severity, duration, and / or frequency. That is, the subject's migraine may have become unresponsive to therapy with a CGRP inhibitor, i.e., the CGRP inhibitor may have lost its effectiveness in the subject.

[0039] The lack of efficacy, partial efficacy, or loss of efficacy of a CGRP inhibitor can be self-diagnosed by the subject or diagnosed by the subject's physician. In the case of non-human subjects, the lack of efficacy or partial efficacy can be diagnosed by the subject's owner, breeder, veterinarian, or someone able to monitor the subject's condition.

[0040] Alternatively, a subject may be an inadequate CGRP responder (aCGRP-IR), also referred to as an inadequate responder to anti-CGRP therapy, in which the subject has failed to respond to three or more adequate doses (for two or more months at the maximum tolerated dose) of small molecule migraine preventative treatments from different classes and has failed one or more aCGRP therapies (aCGRP-inadequate responder [aCGRP-IR]), where failure is defined as lack of clinically meaningful improvement in the treating physician's judgment or discontinuation of aCGRP therapy due to intolerable AEs.

[0041] As demonstrated in the Examples below, the present inventors have found that migraine can be either CGRP-dependent or CGRP-independent. Without being bound by theory, CGRP-dependent migraine is caused by signaling through the CGRP / CGRP receptor axis as described above, whereas CGRP-independent migraine is thought to have an unknown mechanism that does not rely on signaling through the CGRP / CGRP receptor axis. While PAR2 inhibition has previously been shown to be effective in preventing migraine in a mouse model of CGRP-dependent migraine (Kopruszinski et al., Cephalagia 40(14)15-35-1550, 2020), the present inventors are the first to demonstrate that PAR2 inhibition is effective in preventing CGRP-independent migraine.

[0042] Migraine that is refractory (or resistant) to treatment with a CGRP inhibitor may be CGRP-independent migraine. For patients suffering from CGRP-independent migraine, CGRP inhibitor therapy may be predicted to be ineffective because signaling through the CGRP / CGRP receptor axis does not play a role in the pathogenesis of migraine. For such subjects, PAR2 inhibitor therapy is an appropriate and effective alternative. Therefore, the therapy presented herein may be considered a method for treating, alleviating, or preventing CGRP-independent migraine. However, currently, there is no way to determine whether a migraine patient is suffering from CGRP-dependent or CGRP-independent migraine other than administering a CGRP inhibitor to the patient and assessing its effectiveness. Therefore, patients for whom CGRP inhibitors are effective migraine therapy may be considered to be suffering from CGRP-dependent migraine, and patients for whom CGRP inhibitors are ineffective migraine therapy may be considered to be suffering from CGRP-independent migraine.

[0043] In some cases, patients may suffer from both CGRP-dependent migraine and CGRP-independent migraine simultaneously.For such patients, CGRP inhibitors are predicted to be partially effective as migraine therapy, since they block the CGRP-dependent contribution to the patient's migraine, but not the CGRP-independent contribution.As mentioned above, such patients may experience minor or partial improvement in the severity, duration and / or frequency of migraine through therapy with CGRP inhibitors, but the resulting benefit is much less than that of patients whose migraine is purely CGRP-dependent.Such patients may benefit from therapy using the methods presented herein to block the CGRP-independent contribution to migraine.

[0044] Alternatively, a subject may be intolerant to a CGRP inhibitor and therefore unable to undergo CGRP inhibitor therapy, in which case therapy with a PAR2 inhibitor as provided herein provides a suitable alternative to CGRP inhibitor therapy.

[0045] A subject is considered to be intolerant to a CGRP inhibitor if the subject cannot undergo CGRP inhibitor therapy for reasons unrelated to efficacy.That is, a subject who is intolerant to a CGRP inhibitor can be any subject for whom migraine therapy using a CGRP inhibitor is contraindicated for reasons other than lack of efficacy.For example, such a subject may be allergic to a CGRP inhibitor (or may be allergic to one or more of the excipients in the pharmaceutical composition in which the CGRP inhibitor is administered), may have another health condition that makes it unsafe for the subject to take or be administered a CGRP inhibitor, or may take one or more other drugs that are incompatible with a CGRP inhibitor.Intolerance to a specific CGRP inhibitor can be routinely diagnosed by a physician.

[0046] In this case, the subject may be intolerant to one CGRP inhibitor or to multiple CGRP inhibitors (i.e., two or more CGRP inhibitors). For example, the subject may be intolerant to one or more classes of CGRP inhibitors.

[0047] If the subject has tried multiple different CGRP inhibitors, the subject has been found to be non-responsive to treatment with each of the CGRP inhibitors tried and / or to be intolerant to each of the CGRP inhibitors tried.

[0048] As mentioned above, the subject may be any animal suffering from migraine. Thus, the animal may be human or non-human. In the methods provided herein, the subject is either non-responsive to therapy using an inhibitor of CGRP of the subject's species or intolerant to inhibitors of CGRP of the subject's species. For example, the human subject treated as described herein is either non-responsive to therapy using an inhibitor of human CGRP or intolerant to inhibitors of human CGRP (e.g., allergic) for migraine therapy.

[0049] As mentioned above, currently, there are first-line and second-line migraine treatments that are generally prescribed before CGRP inhibitors, with CGRP inhibitors usually being considered third-line treatments. A subject treated according to the methods provided herein may also be non-responsive to therapy with a first-line migraine treatment or a second-line migraine treatment (in addition to being non-responsive to or intolerant of treatment with a CGRP inhibitor). Thus, a subject may be non-responsive to therapy with a first-line migraine treatment. A subject may be non-responsive to therapy with a second-line migraine treatment. A subject may be non-responsive to therapy with a first-line migraine treatment and non-responsive to therapy with a second-line migraine treatment.

[0050] First-line migraine treatments include triptans, beta-blockers, topiramate (a carbonic anhydrase inhibitor), and candesartan (an angiotensin receptor blocker). Examples of triptans that can be used as first-line migraine treatments include zolmitriptan, sumatriptan, rizatriptan, almotriptan, eletriptan, frovatriptan, and naratriptan. The subject may be non-responsive to migraine therapy with one or more triptans, for example, one or more of the triptans listed above. Examples of beta-blockers that can be used as first-line migraine treatments include atenolol, metoprolol, timolol, nadolol, and propranolol. The subject may be non-responsive to migraine therapy with one or more beta-blockers, for example, one or more of the beta-blockers listed above. The subject may be non-responsive to migraine therapy with candesartan. The subject may be non-responsive to topiramate therapy.

[0051] A subject may be non-responsive to migraine therapy with one first-line treatment (as described above) or multiple first-line treatments. For example, a subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with one or more beta-blockers. A subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with candesartan. A subject may be non-responsive to migraine therapy with one or more beta-blockers and non-responsive to migraine therapy with candesartan. A subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with topiramate. A subject may be non-responsive to migraine therapy with one or more beta-blockers and non-responsive to migraine therapy with topiramate. A subject may be non-responsive to migraine therapy with topiramate and non-responsive to migraine therapy with candesartan. The subject may be non-responsive to migraine therapy with one or more triptans, non-responsive to migraine therapy with one or more beta-blockers, and non-responsive to migraine therapy with candesartan. The subject may be non-responsive to migraine therapy with one or more triptans, non-responsive to migraine therapy with one or more beta-blockers, and non-responsive to migraine therapy with topiramate. The subject may be non-responsive to migraine therapy with one or more triptans, non-responsive to migraine therapy with topiramate blockers, and non-responsive to migraine therapy with candesartan. The subject may be non-responsive to migraine therapy with one or more beta-blockers, non-responsive to migraine therapy with topiramate, and non-responsive to migraine therapy with candesartan. The subject may be non-responsive to migraine therapy with one or more beta-blockers, non-responsive to migraine therapy with one or more triptans, non-responsive to migraine therapy with topiramate, and non-responsive to migraine therapy with candesartan.That is, the subject may be non-responsive to migraine therapy with one or more triptans, one or more beta-blockers, topiramate, and / or candesartan.

[0052] Second-line migraine treatments include calcium channel blockers, tricyclic antidepressants, and valproic acid and its salts. Examples of calcium channel blockers that can be used as second-line migraine treatments include verapamil, nimodipine, nifedipine, nicardipine, and flunarizine. The subject may be non-responsive to migraine therapy with one or more calcium channel blockers, such as one or more of the calcium channel blockers listed above. Examples of tricyclic antidepressants that can be used as second-line migraine treatments include amitriptyline, nortriptyline, clomipramine, and opipramol. The subject may be non-responsive to migraine therapy with one or more tricyclic antidepressants, such as one or more of the tricyclic antidepressants listed above. Examples of salts of valproic acid that can be used as second-line migraine treatments include sodium valproate. The subject may be non-responsive to migraine therapy with valproic acid or its salts, such as sodium valproate.

[0053] The subject may be non-responsive to migraine therapy with one second-line treatment (as described above) or multiple second-line treatments. For example, the subject may be non-responsive to migraine therapy with one or more calcium channel blockers and non-responsive to migraine therapy with one or more tricyclic antidepressants. The subject may be non-responsive to migraine therapy with one or more calcium channel blockers and non-responsive to migraine therapy with valproic acid or a salt thereof (e.g., sodium valproate). The subject may be non-responsive to migraine therapy with one or more tricyclic antidepressants and non-responsive to migraine therapy with valproic acid or a salt thereof (e.g., sodium valproate). The subject may be non-responsive to migraine therapy with one or more tricyclic antidepressants, non-responsive to migraine therapy with one or more calcium channel blockers, and non-responsive to migraine therapy with valproic acid or a salt thereof (e.g., sodium valproate). That is, the subject may be non-responsive to migraine therapy with one or more calcium channel blockers, one or more tricyclic antidepressants, and / or valproic acid or a salt thereof (eg, sodium valproate).

[0054] Thus, a subject may be non-responsive to migraine therapy with a first-line migraine treatment and / or a second-line migraine treatment. A subject may be non-responsive to one or more of the first-line migraine treatments described above, and / or may be non-responsive to one or more second-line migraine treatments as described above. A subject may be non-responsive to migraine therapy with one or more triptans, one or more beta-blockers, topiramate, candesartan, one or more calcium channel blockers, one or more tricyclic antidepressants, and / or valproic acid or a salt thereof (e.g., sodium valproate).

[0055] Thus, a subject treated according to the methods provided herein may have previously been treated with at least one first-line migraine therapy and / or at least one second-line migraine therapy, where the treatment with the first and / or second migraine therapy was unsuccessful. That is, the first-line migraine therapy and / or second-line migraine therapy did not effectively treat, alleviate, or prevent the subject's migraine. Thus, "non-responsive" to therapy with a first-line migraine therapy and / or second-line migraine therapy has an equivalent or corresponding meaning to non-responsiveness to therapy with a CGRP inhibitor, i.e., treatment of the subject with the first-line migraine therapy and / or second-line migraine therapy lacked efficacy, was only partially effective, or lost efficacy.

[0056] Additionally or alternatively, the subject may be intolerant to the first-line migraine therapy and / or second-line migraine therapy described above. "Intolerance" of a first-line migraine therapy or a second-line migraine therapy has a meaning equivalent to or corresponding to intolerance to a CGRP inhibitor described above, i.e., the subject is unable to undergo treatment with that first-line migraine therapy or second-line migraine therapy for reasons unrelated to efficacy, e.g., for reasons described above in connection with intolerance to CGRP inhibitors.

[0057] If the subject has tried more than one first-line migraine therapy or second-line migraine therapy, the subject is found to be non-responsive to treatment with each of the first-line migraine therapies or second-line migraine therapies attempted or to be intolerant to each of the first-line migraine therapies or second-line migraine therapies attempted.

[0058] The subject may be a subject who has been or is overusing acute medications in an attempt to control the migraine condition. "Acute medications" include over-the-counter analgesics, such as paracetamol and ibuprofen, as well as first- and second-line treatments (e.g., triptans) that are taken in response to migraine symptoms rather than according to a prescribed dosing schedule. "Overuse" of such acute medications may mean that the subject has taken or is taking more than the maximum recommended dose of the acute medication, or that the subject is using the acute medication too frequently. If the subject is taking the acute medication at a dose that causes side effects or impairs (or is at risk of impairing) the subject's overall health or well-being, such use is considered overuse.

[0059] CGRP inhibitors Most commonly, the subject to be treated is a human, and therefore the CGRP inhibitor is a human CGRP inhibitor. That is, the subject may be non-responsive to migraine therapy using a human CGRP inhibitor or intolerant to a human CGRP inhibitor. As described above, a CGRP inhibitor as defined herein is an agent that blocks the activity of CGRP, in particular, prevents CGRP from activating CGRP receptors.

[0060] Human CGRP has two distinct forms: αCGRP (SEQ ID NO: 12) and βCGRP (SEQ ID NO: 13). αCGRP is encoded by the CALCA gene, and βCGRP is encoded by the CALCB gene. The same may be true for other animals. The two forms of human CGRP share over 90% sequence identity, differ by only three amino acids, and have essentially equivalent biological activity; however, αCGRP is the predominant form found in the central and peripheral nervous systems, while βCGRP is found primarily in the enteric nervous system (Russell et al., Physiological Reviews 94(4):1099-1142, 2014). The αCGRP and βCGRP peptides are collectively referred to as CGRP. Both αCGRP and βCGRP signal through the same CGRP receptor. The CGRP inhibitors defined herein may be inhibitors of αCGRP (particularly human αCGRP) or βCGRP (particularly human βCGRP), but are preferably and generally inhibitors of both αCGRP and βCGRP. That is, the CGRP inhibitors defined herein may prevent αCGRP from activating the CGRP receptor, or may prevent βCGRP from activating the CGRP receptor, but are preferably and generally inhibitors of both αCGRP and βCGRP from activating the CGRP receptor. The CGRP receptor includes two proteins: CLR and RAMP1. The human CLR protein has UniProt accession number Q16602, and the human RAMP1 protein has UniProt accession number O60894.

[0061] The CGRP inhibitor to which a subject is intolerant and / or non-responsive can be an antibody. In this case, the antibody prevents CGRP from binding to and / or activating the CGRP receptor. Generally, such an antibody binds to either CGRP or the CGRP receptor. When the antibody binds to the CGRP receptor, it binds to either the CLR protein or the RAMP1 protein.

[0062] Antibodies that are CGRP inhibitors can prevent CGRP from activating the CGRP receptor by blocking the binding of CGRP to the CGRP receptor. Such antibodies can act sterically by binding to CGRP at or near the position where CGRP binds to the CGRP receptor, or by binding to the CGRP receptor at or near the position where the CGRP receptor binds to CGRP, so as to physically block the CGRP-CGRP receptor interaction. Alternatively, the binding of CGRP to the CGRP receptor can be inhibited by antibodies that bind to CGRP or the CGRP receptor at a site distant from the CGRP-CGRP receptor interface but cause a change in the shape or conformation of CGRP or the CGRP receptor, thereby inhibiting the binding of CGRP to the CGRP receptor.

[0063] Alternatively, an antibody that is a CGRP inhibitor may not prevent CGRP from binding to the CGRP receptor, but instead inhibits activation of the CGRP receptor in response to CGRP binding. For example, the antibody may bind to the CGRP receptor and lock it in an inactive conformation, thereby preventing activation of the receptor by CGRP binding.

[0064] Known therapeutic antibodies that act as CGRP inhibitors include erenumab (Amgen / Novartis) that binds to CGRP receptor; fremanezumab (Teva) that binds to CGRP; galcanezumab (Eli Lilly) that binds to CGRP; and eptinezumab (Lundbeck) that binds to CGRP. The subject treated according to the method herein may be unresponsive to migraine treatment with an antibody that inhibits CGRP, particularly an antibody that binds to CGRP or CGRP receptor and prevents CGRP from binding to CGRP receptor or prevents the activation of CGRP receptor, or may be intolerant to the antibody. For example, the subject may be unresponsive to migraine treatment with erenumab, fremanezumab, galcanezumab and / or eptinezumab, or may be intolerant to erenumab, fremanezumab, galcanezumab and / or eptinezumab.

[0065] Alternatively, the CGRP inhibitor to which the subject is intolerant and / or non-responsive may be a small molecule. Small molecule inhibitors of CGRP may act to prevent CGRP from activating the CGRP receptor in any of the ways described above for CGRP inhibitor antibodies. Generally, small molecule inhibitors of CGRP bind to either CGRP or the CGRP receptor and act directly or indirectly to block the binding of CGRP to the CGRP receptor, or they do not inhibit the binding of CGRP to the CGRP receptor but inhibit the activation of the CGRP receptor in response to the binding of CGRP to the CGRP receptor.

[0066] Known small molecules that act as CGRP inhibitors include gepants, which bind to CGRP receptors and inhibit their activation. Known gepants include rimegepant, ubrogepant, zabegepant, olcegepant, and atogepant. The subject treated according to the method herein may be non-responsive to migraine treatment with one or more gepants, or intolerant to one or more gepants. For example, the subject may be non-responsive to migraine treatment with rimegepant, ubrogepant, zabegepant, olcegepant, and / or atogepant, or intolerant to rimegepant, ubrogepant, zabegepant, olcegepant, and / or atogepant. In particular, subjects treated according to the methods herein may be non-responsive to migraine treatment with gepants or may be intolerant to gepants, i.e., subjects treated according to the methods herein may be non-responsive to migraine treatment with small molecules of the gepant class or may be intolerant to small molecules of the gepant class.

[0067] A subject may be non-responsive to migraine treatment with any other therapy or type of therapy that targets the CGRP-CGRP receptor signaling axis, or may be intolerant to any other therapy or type of therapy that targets the CGRP-CGRP receptor signaling axis. For example, a subject may be non-responsive to migraine treatment with large molecule therapeutics that inhibit CGRP, e.g., peptide or nucleic acid drugs that inhibit CGRP by the mechanisms described above for antibodies and small molecules, or gene therapy that acts to inhibit CGRP activity (e.g., by knocking out or knocking down CGRP or the CGRP receptor), or may be intolerant to large molecule therapeutics that inhibit CGRP, e.g., peptide or nucleic acid drugs that inhibit CGRP by the mechanisms described above for antibodies and small molecules, or gene therapy that acts to inhibit CGRP activity (e.g., by knocking out or knocking down CGRP or the CGRP receptor).

[0068] A subject may be non-responsive to migraine therapy with one or more classes of CGRP inhibitors and / or may be intolerant to one or more classes of CGRP inhibitors. For example, a subject may be non-responsive to migraine therapy with both an antibody and a small molecule that inhibits CGRP and / or may be intolerant to both an antibody and a small molecule that inhibits CGRP, particularly, a subject may be non-responsive to migraine therapy with an antibody and a gepant that inhibits CGRP and / or may be intolerant to an antibody and a gepant that inhibits CGRP.

[0069] Antibodies against PAR2 In the methods provided herein, an antibody or antigen-binding fragment thereof that specifically binds to PAR2 is administered to a subject.

[0070] As defined herein, and in accordance with standard terminology in the art, an antibody is a protein composed of two heavy chains and two light chains. The light chains are shorter (and therefore lighter) than the heavy chains. The heavy chains contain an N-terminal heavy chain variable domain (V H ), and the light chain comprises an N-terminal light chain variable domain (V L ) Each chain contains a constant domain C-terminal to the variable domain.

[0071] The specificity of an antibody is determined by the sequence of its variable region. Both the light and heavy chains of an antibody contain three hypervariable complementarity determining regions (CDRs), such as those described herein below. In a light and heavy chain pair, the CDRs of the two chains form an antigen-binding site. The CDR sequences determine the specificity of the antibody. The three CDRs of a heavy chain are known from N- to C-terminus as VHCDR1, VHCDR2, and VHCDR3, and the three CDRs of a light chain are known from N- to C-terminus as VLCDR1, VLCDR2, and VLCDR3. Framework regions are located between the CDRs and between the CDRs and the end of the variable domain. An antigen-binding fragment of an antibody is a fragment or synthetic construct that contains one or more antigen-binding sites of an antibody but is not the whole antibody. Generally, an antigen-binding fragment of an antibody comprises V L and VH It contains the entire domain sequence but lacks the heavy and light chain constant domains and does not contain the entire heavy and light chain constant domains.

[0072] As described above, the antibody (or fragment thereof) for use in the methods provided herein specifically binds to PAR2. The antibody or fragment thereof may bind to PAR2 from any species of interest. In particular, the antibody or fragment thereof binds to PAR2 from the species to which the subject belongs. Thus, the antibody or fragment thereof may bind to PAR2 from, for example, canines, felines, or equines. However, most commonly, the antibody or fragment thereof specifically binds to human PAR2. The amino acid sequence of human PAR2 has UniProt accession number P55085 and is also set forth in SEQ ID NO: 14. When an antibody used herein specifically binds to human PAR2, the antibody may bind to human PAR2 but not to PAR2 from other non-human species, or may bind to human PAR2 and PAR2 from other non-human species.

[0073] An antibody that specifically binds to human PAR2 is an antibody that binds to human PAR2 with greater affinity than it binds to other molecules (e.g., the affinities described below), or at least with higher affinity than the majority of other molecules. Thus, for example, when an antibody that binds to human PAR2 is contacted with a human cell lysate, the antibody will primarily bind to PAR2. In particular, the antibody will bind to a sequence or conformation present in human PAR2, preferably a unique sequence or conformation not present in other molecules. An antibody that specifically binds to human PAR2 does not necessarily bind exclusively to human PAR2; the antibody may cross-react with certain other undefined target molecules or may exhibit a level of non-specific binding when contacted with a mixture of multiple molecules (e.g., cell lysate or the like). In particular, an antibody that specifically binds to human PAR2 may exhibit cross-reactivity with PAR2 from other species, such as those listed above. Nevertheless, one skilled in the art can easily determine whether an antibody or a fragment thereof specifically binds to human PAR2 using standard techniques in the art, such as ELISA, Western blot, surface plasmon resonance (SPR), etc.

[0074] As further described below, the antibodies or fragments thereof for use herein may bind to PAR2 in a pH-dependent manner. Anti-PAR2 antibodies or antigen-binding fragments thereof for use herein have high affinity for PAR2, e.g., a K of less than about 5 nM, less than 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 650 pM, less than 600 pM, less than 500 pM, less than 200 pM, less than 100 pM, or less than 50 pM. DPreferably, high affinity binding occurs at physiological, extracellular pH (i.e., about pH 7.4). The antibodies or antigen-binding fragments thereof for use herein may bind to PAR2 with slightly lower affinity at slightly acidic pH (e.g., pH 6.0) compared to physiological, extracellular pH. That is, the antibodies or fragments thereof may bind to PAR2 with higher (or greater) affinity at pH 7.4 than at pH 6.0. For example, at about pH 6.0, the antibodies or fragments thereof for use herein may bind to PAR2 with a K of about greater than 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, or 100 nM. D The K at which an antibody or fragment thereof binds to PAR2 at any given pH D can be determined using standard methods in the art, such as SPR. Anti-PAR2 antibodies or antigen-binding fragments thereof for use herein bind to PAR2 at pH 7.4 and at a temperature of, for example, 25° C. or 37° C., with a dissociation half-life (t) of greater than about 1.5 minutes, greater than 1.75 minutes, greater than 2 minutes, greater than 2.5 minutes, greater than 3 minutes, greater than 5 minutes, greater than 10 minutes, greater than 20 minutes, or greater than 30 minutes, as measured using an assay such as surface plasmon resonance. 1 / 2 The anti-PAR2 antibody or antigen-binding fragment thereof may specifically bind to PAR2 with a dissociation half-life (t) of less than about 1 minute, less than 45 seconds, less than 30 seconds, less than 20 seconds, less than 15 seconds, less than 13 seconds, less than 7 seconds, less than 5 seconds, or less than 3 seconds at a slightly acidic pH (e.g., pH 6) and a temperature of, for example, 25°C or 37°C, as measured using an assay such as surface plasmon resonance. 1 / 2 ) can be bonded.

[0075] The antibodies or fragments thereof for use in the methods provided herein inhibit the activity of PAR2. That is, the antibodies or fragments thereof used herein may be referred to as neutralizing anti-PAR2 antibodies or antagonistic anti-PAR2 antibodies. A neutralizing anti-PAR2 antibody may neutralize PAR2 activity, for example, by (i) interfering with the interaction of PAR2 with a protease (e.g., trypsin, tryptase, and / or matriptase); (ii) inhibiting cleavage of PAR2 by a protease; or (iii) inhibiting PAR2 signaling or PAR2 activation. For example, the antibody or antigen-binding fragment thereof may inhibit the conversion of inactive, uncleaved PAR2 to active, cleaved PAR2 and / or inhibit the exposure of a tethered ligand. Alternatively, the antibody or antigen-binding fragment thereof may inhibit the activation of PAR2 by a tethered ligand of PAR2, for example, by inhibiting the binding of the tethered ligand to the second transmembrane domain of PAR2 (which, as described above, is required for PAR2 activation).

[0076] Therefore, the antibody or fragment thereof for use in the methods provided herein can inhibit PAR2 activity by inhibiting cleavage of the PAR2 extracellular domain. As described above, cleavage of the PAR2 extracellular domain by serine proteases leads to PAR2 activation, and therefore, preventing cleavage of this domain is useful for inhibiting PAR2 activation. Thus, the antibody (or fragment thereof) can prevent proteolytic cleavage of the PAR2 extracellular domain by serine proteases. In particular, the antibody (or fragment thereof) can prevent cleavage of the PAR2 extracellular domain by trypsin, tryptase, or matriptase. Other proteases that can act to cleave the PAR2 extracellular domain include elastase and proteinase 3. The antibody (or fragment thereof) can inhibit PAR2 cleavage by any of these enzymes, as well as by any other protease enzyme capable of cleaving PAR2. An antibody can inhibit PAR2 cleavage, for example, by binding to PAR2 at or near the cleavage site, thereby blocking protease access to the cleavage site. Thus, an antibody or antigen-binding fragment thereof can prevent proteases from interacting with PAR2, particularly trypsin, tryptase, and / or matriptase, from interacting with PAR2, thereby inhibiting cleavage and the resulting activation of PAR2. Thus, an antibody or fragment thereof for use herein can bind to the intact (uncleaved) extracellular domain of PAR2, i.e., the antibody or fragment thereof can bind to an epitope located in the extracellular domain of PAR2 before cleavage (such an epitope can also be present in the extracellular domain of PAR2 after cleavage). An important feature of such an epitope located in the PAR2 extracellular domain before cleavage is that antibody binding to the epitope inhibits proteolytic cleavage of the PAR2 extracellular domain, e.g., by blocking protease access to the PAR2 cleavage site.

[0077] The anti-PAR2 antibody or fragment thereof has an IC of less than about 15 nM, as measured in vitro by a binding assay of PAR2 interaction with a protease (e.g., trypsin), such as that described in WO2018 / 167322 (hereby incorporated by reference). 50 The antibody or antigen-binding fragment may block the interaction of PAR2 with a protease (e.g., trypsin) in vitro at a pH of about 7.4 with an IC of less than about 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 500 pM, less than 400 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 5 pM, less than 1 pM, or less than 0.1 pM. 50 The antibody or fragment thereof may block the interaction of PAR2 with a protease (e.g., trypsin) in vitro at a pH of about 6.0 with an IC of greater than about 300 nM, greater than 500 nM, greater than 750 nM, greater than 1000 nM, greater than 1100 nM, or greater than 1200 nM. 50 It can be cut off by a value.

[0078] As described above, cleavage of the PAR2 extracellular domain exposes a PAR2 tethered ligand, which is a PAR2 sequence that binds to the PAR2 activation site, thereby activating the receptor. Thus, the antibody or fragment thereof for use herein can inhibit PAR2 activity by inhibiting the exposure of the PAR2 tethered ligand or by preventing the PAR2 tethered ligand from interacting with the PAR2 activation site. The PAR2 activation site is located within the extracellular loop 2 domain of PAR2, and therefore, the antibody or fragment thereof can prevent the PAR2 tethered ligand from interacting with the extracellular loop 2 domain of PAR2.

[0079] Inhibition of exposure of the PAR2 tethered ligand or the interaction of the PAR2 tethered ligand with the extracellular loop 2 domain of PAR2 can, of course, be achieved by first preventing PAR2 cleavage, as described above. Alternatively, the antibody may block exposure of the tethered ligand or the interaction of the tethered ligand with the PAR2 activation site after cleavage of the PAR2 extracellular domain. In this case, the antibody or a fragment thereof may bind to the PAR2 tethered ligand, thereby blocking exposure of the PAR2 tethered ligand and preventing the PAR2 tethered ligand from interacting with the PAR2 activation site. The human PAR2 tethered ligand has the amino acid sequence set forth in SEQ ID NO: 15. The antibody or fragment thereof used herein may bind to PAR2 at an epitope within SEQ ID NO: 15 or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 15.

[0080] To inhibit the interaction of a PAR2 tethered ligand with a PAR2 activation site, an antibody or fragment thereof for use herein can bind to the PAR2 activation site, thereby blocking the interaction between the activation site and the tethered ligand. As described above, the PAR2 activation site is located within the extracellular loop 2 domain of PAR2 (i.e., the extracellular loop between transmembrane helices 2 and 3 of PAR2). Therefore, an antibody or fragment thereof can bind to the extracellular loop 2 domain of PAR2. The extracellular loop 2 domain of PAR2 is located at residues 138-149 of PAR2 (SEQ ID NO: 16). An antibody or fragment thereof for use herein can bind to PAR2 at an epitope within SEQ ID NO: 16 or an amino acid sequence having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 16.

[0081] The antibodies or fragments thereof for use herein inhibit the activity of PAR2. This inhibition can be considered as inhibition of PAR2 activation. As described above, this inhibition can be achieved by any mechanism that is effective in inhibiting PAR2 activity. The inhibition caused by the anti-PAR2 antibodies or fragments thereof for use herein can be complete (i.e., binding of the antibody to PAR2 completely inhibits PAR2 activity, thus completely inactivating the receptor). However, inhibition need not be complete, so long as it is detectable using a suitable assay and is sufficient to achieve successful treatment, alleviation, or prevention of migraine in the subject to which the antibody or antigen-binding fragment thereof is administered. For example, the antibody or antigen-binding fragment thereof can inhibit PAR2 activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to uninhibited active PAR2. A higher degree of inhibition is advantageous because an antibody with a higher degree of inhibition may achieve better therapeutic results than an antibody with a lower inhibitory effect, or may achieve the same results at a lower dose.

[0082] Examples of several assays for detecting the activity of anti-PAR2 antibodies or antigen-binding fragments thereof in inhibiting PAR2 are described in the Examples of WO2018 / 167322. For example, PAR2 inhibition can be measured using a cell-based assay, particularly a calcium flux assay. PAR2 activation increases intracellular Ca 2+ Increase in concentration (resulting in Ca from outside the cell into the cell) 2+ Intracellular Ca influx is not triggered. 2+ It is believed that intracellular Ca stores are mobilized. 2+ Changes in concentration can be measured as a proxy for PAR2 activation. Cells that endogenously express PAR2 (e.g., human A549 cells, cynomolgus monkey CYNOM-K1 cells, rat KNRK cells, or mouse LL / 2 cells) can be used in such assays.

[0083] In a cell-based calcium flux assay for measuring antibody-mediated PAR2 inhibition, PAR2-expressing cells are first loaded with a calcium dye (e.g., ScreenQuest™ Fluo-8 dye, AAT Bioquest, USA) and then pretreated with the antibody or antibody fragment of interest. If necessary, the cells can also be pretreated with thrombin to desensitize PAR1 activity. Trypsin is then applied to the cells, and the calcium response can be measured based on the activity of the calcium dye. For example, Fluo-8 dye, described above and exemplified in WO2018 / 167322, is a fluorescent dye whose fluorescence increases upon calcium binding. The change in fluorescence in response to the application of trypsin to the cells indicates the degree of PAR2 activity caused by trypsin. Those skilled in the art will be aware of additional anti-PAR2 antibody activity assays that can be used to assess PAR2 inhibition.

[0084] The anti-PAR2 antibodies or fragments thereof used herein may cause at least a 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or even a 100% reduction in the trypsin response by PAR2-expressing cells in the calcium flux assay described above. The antibodies or antigen-binding fragments thereof used herein may cause calcium influx with an IC of less than 1 nM, less than 500 pM, less than 400 pM, less than 200 pM, less than 100 pM, less than 50 pM, less than 10 pM, less than 5 pM, less than 1 pM, or less than 0.1 pM in a calcium flux assay (e.g., as described above). 50 can be inhibited by

[0085] The antibodies or antigen-binding fragments thereof used herein have a calcium flux of 5.0 x 10 in a calcium flux assay in human A549 cells. -10 Less than M, 4.5 x 10 -10 Less than M, 4.0 x 10 -10 Less than M, 3.5 x 10 -10 , 3.0×10 -10 Less than M, 2.5 x 10 -10 Less than M, 2.0 x 10-10 Less than M, 1.5 x 10 -10 Less than M or 1.0 x 10 -10 IC less than M 50 The antibodies or antigen-binding fragments thereof used herein may have a calcium flux assay in rat KNRK cells of 10 -9 Less than M, 9.5 x 10 -10 Less than M, 9.0 x 10 -10 Less than M, 8.5 x 10 -10 Less than M, 8 x 10 -10 Less than M, 7.5 x 10 -10 Less than M, 7.0 x 10 -10 Less than M, 6.5 x 10 -10 Less than M, 6.0 x 10 -10 Less than M, 5.5 x 10 -10 Less than M or 5 x 10 -10 IC less than M 50 The antibodies or antigen-binding fragments thereof used herein may have a calcium flux of 10 or more in a calcium flux assay in cynomolgus monkey CYNOM-K1 cells. -10 Less than M, 9.5 x 10 -11 Less than M, 9.0 x 10 -11 Less than M, 8.5 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7.5 x 10 -11 Less than M, 7.0 x 10 -11 Less than M, 6.5 x 10 -11 Less than M, 6.0 x 10 -11 Less than M, 5.5 x 10 -11 Less than M or 5.0 x 10 -11 IC less than M 50 The antibodies or antigen-binding fragments thereof used herein may have a calcium influx assay in mouse LL / 2 cells of 10 -10 Less than M, 9.5 x 10 -11 Less than M, 9.0 x 10 -11 Less than M, 8.5 x 10 -11 Less than M, 8 x 10 -11 Less than M, 7.5 x 10 -11 Less than M, 7.0 x 10 -11 Less than M, 6.5 x 10 -11 Less than M, 6.0 x 10 -11 Less than M, 5.5 x 10-11 Less than M, 5.0 x 10 -11 Less than M, 4.5 x 10 -11 Less than M or 4.0 x 10 -11 IC less than M 50 may have:

[0086] As noted above, antibodies or antigen-binding fragments thereof (also referred to herein as "antibody fragments") are used in the methods provided herein. An "antibody" is an immunoglobulin having the characteristics described herein above.

[0087] As used herein, antibodies or antibody fragments are generally monoclonal, i.e., monoclonal antibodies or monoclonal antibody fragments can be used. "Monoclonal antibody" refers to an antibody preparation consisting of a single antibody species, i.e., all antibodies in the preparation have the same amino acid sequence, including the same CDRs, and therefore bind to the same epitope of their target antigen ("target antigen" refers to an antigen containing the epitope to which a particular antibody binds; i.e., the target antigen of an anti-PAR2 antibody is PAR2), and have the same effect. Thus, a monoclonal antibody fragment refers to a preparation of antibody fragments consisting of a single species of antibody fragment. In other words, as used herein, an antibody or a fragment thereof is generally not used in the context of or as part of a polyclonal mixture of antibodies or antibody fragments.

[0088] In the above-described antibodies, the CDR sequences are located in the heavy and light chain variable domains. The CDR sequences are placed within a polypeptide framework that appropriately positions the CDRs for antigen binding. Thus, the remaining portions of the variable domains (i.e., the portions of the variable domain sequence that do not form part of any one of the CDRs) constitute the framework regions. The N-terminus of the mature variable domain forms framework region 1 (FR1), the polypeptide sequence between CDR1 and CDR2 forms FR2, the polypeptide sequence between CDR2 and CDR3 forms FR3, and the polypeptide sequence connecting CDR3 to the constant domain forms FR4. The variable region, including the CDR and framework regions, of the antibody (or fragment thereof) used herein may have any appropriate sequence that achieves binding to and inhibition of PAR2. The antibody or antigen-binding fragment thereof used herein may have at least one variable domain (e.g., V H and / or V L Generally, antibodies or antigen-binding fragments thereof contain V H and V L Although certain antibodies or fragments thereof contain a single variable domain, particularly a V H Only the domain may be required, for example a camelid single domain antibody may be used.

[0089] As is well known in the art, antibodies can belong to several different isotypes. The isotype of an antibody is determined by the sequence of its constant region. In humans, antibody isotypes are IgG, IgE, IgM, IgA, and IgD. Some isotypes can be further divided into subtypes; for example, IgG antibodies have four subtypes: IgG1, IgG2, IgG3, and IgG4. Antibodies (or antibody fragments containing a portion of the constant region) are used in the methods herein, and the antibody may be of any isotype, i.e., IgG, IgE, IgM, IgA, or IgD antibodies. In particular, the antibody may be an IgG antibody. When an IgG antibody is used, it may be of any subtype, i.e., IgG1, IgG2, IgG3, or IgG4 antibodies. In particular, the antibody may be an IgG1 antibody. The antibody used herein may contain a κ or λ light chain.

[0090] The antibody or fragment thereof used in the methods herein may be a multispecific, e.g., bispecific, monoclonal antibody. A multispecific antibody contains regions or domains (antigen-binding regions) that bind to at least two different molecular binding partners, e.g., two or more different antigens or epitopes. In the case of a bispecific antibody, the antibody has the above-described structure, except that the variable domains of the two heavy chains and the two light chains are different, thus forming two different antigen-binding regions. In a multispecific (e.g., bispecific) antibody for use herein, one of the antigen-binding regions has the CDR sequence of an antagonist anti-PAR2 antibody. The other antigen-binding region of the multispecific antibody is different and therefore binds to PAR2 and does not act to antagonize it. Generally, the other antigen-binding region of a multispecific antibody for use herein binds to a different target, i.e., binds to a target other than PAR2.

[0091] As mentioned above, antigen-binding fragments of antibodies can be used herein. Antibody fragments are discussed in Rodrigo et al., Antibodies, Vol. 4 (3), pp. 259-277, 2015. Antibody fragments that can be used herein include, for example, Fab, F(ab')2, Fab', and Fv fragments. Fab fragments are discussed in Roitt et al., Immunology second edition (1989), Churchill Livingstone, London. Fab fragments consist of the antigen-binding domain of an antibody; that is, an individual antibody can be considered to contain two Fab fragments, each consisting of a light chain and the N-terminal section of a heavy chain linked to the light chain. Thus, a Fab fragment consists of the entire light chain and the V domain of the heavy chain linked to it. H and C H Fab fragments contain 1 domain. Fab fragments can be obtained by digesting an antibody with papain.

[0092] An F(ab')2 fragment consists of two Fab fragments of an antibody, plus the hinge region of the heavy chain domain containing the disulfide bond that links the two heavy chains together. In other words, an F(ab')2 fragment can be considered as two Fab fragments covalently joined together. An F(ab')2 fragment can be obtained by digesting an antibody with pepsin.

[0093] Reduction of the F(ab')2 fragment yields two Fab' fragments, which can be thought of as Fab fragments containing additional sulfhydryl groups that may be useful for conjugating the fragment to other molecules.

[0094] Fv fragments consist solely of the variable domains of the light and heavy chains. These variable domains are not covalently linked, but are held together only by weak non-covalent interactions. Fv fragments can be engineered to produce synthetic constructs known as single-chain Fv (scFv) molecules. Such engineering is typically achieved by recombinantly engineering a single polypeptide into a V H Domain and V LThis is accomplished by engineering antibody genes to produce a fusion protein containing both the V and V domains. scFv fragments generally contain the V domain, which contributes to the stability of the molecule. H Area and V L The present invention also includes a peptide linker that covalently joins the domains. The linker may contain 1 to 20 amino acids, e.g., 1, 2, 3, or 4 amino acids, 5, 10, or 15 amino acids, or any other convenient intermediate number of amino acids within the range of 1 to 20. The peptide linker may be formed from any generally convenient amino acid residue, e.g., glycine and / or serine. One example of a suitable linker is Gly4Ser. Multimers of such linkers, e.g., dimers, trimers, tetramers, or pentamers, e.g., (Gly4Ser)2, (Gly4Ser)3, (Gly4Ser)4, or (Gly4Ser)5, may be used. However, the presence of a linker is not essential, and V L Domains and V H The domains may be linked by peptide bonds. As used herein, an scFv is defined as an antibody fragment, or an antigen-binding fragment of an antibody.

[0095] As used herein, an antibody fragment may be an analog of an scFv. For example, an scFv can be linked to other antibodies or fragments thereof (e.g., other scFvs, Fab antibody fragments, and antibodies against different targets). An scFv can be linked to other scFvs to form multimers, such as dimers, trimers, or tetramers, which are multispecific scFvs. Bispecific scFvs are sometimes referred to as diabodies, trispecific scFvs are referred to as triabodies, and tetraspecific scFvs are referred to as tetrabodies. Alternatively, an scFv can be linked to other identical scFv molecules, thus forming monospecific but multivalent multimers, such as bivalent dimers or trivalent trimers.

[0096] Antibodies for use in the methods herein may be human antibodies (or fragments thereof) or humanized antibodies (or fragments thereof). Human and humanized antibodies may be advantageously used, particularly for the treatment of human subjects. Thus, the subject treated according to the provided methods may be a human, and the antibody or fragment thereof used to treat the subject may be a human antibody or fragment thereof or a humanized antibody or fragment thereof.

[0097] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid sequences not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Such antibodies are referred to herein as "humanized," as further described below. Similarly, antigen-binding fragments of human antibodies are antibody fragments (as described above) in which any portion of the variable and, if present, constant domains, present in the fragment, are derived from human germline immunoglobulin sequences.

[0098] Antibodies for use in the methods provided herein may be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. Such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, in vivo somatic mutagenesis) to thereby determine the V and C regions of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V H Sequence and V L It may be a sequence that, while derived from and related to a sequence, may not naturally exist within the human antibody germline repertoire in vivo.

[0099] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, with the dimer held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not linked by the interchain disulfide bond, forming a molecule of approximately 75-80 kDa composed of covalently coupled light and heavy chains (half antibody). These forms are extremely difficult to separate, even after affinity purification.

[0100] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed when using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). Antibodies for use herein may have one or more mutations in the hinge, CH2, or CH3 regions (compared to human germline sequences), which may be desirable, for example, to improve the yield of the desired antibody form during production.

[0101] The anti-PAR2 antibodies or antigen-binding fragments thereof for use herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody is derived.

[0102] Human antibodies can be obtained by generating antibodies in a conventional manner by immunizing transgenic mice carrying human immunoglobulin genes with the target antigen (i.e., PAR2). Alternatively, human antibodies can be obtained by using combinatorial libraries to screen for antibodies with the desired activity. For example, various methods for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics are known in the art. Such methods are generally described in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001). For example, one method for generating antibodies of interest is through the use of phage antibody libraries, as described in Lee et al., J. Mol. Biol. (2004), 340(5):1073-93. In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are then sorted by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution. Antibodies for use in the methods presented herein can be obtained by designing an appropriate antigen screening procedure to select phage clones of interest, and then constructing full-length antibody clones using the Fv sequences from the phage clones of interest and appropriate constant region (Fc) sequences.

[0103] "Humanized" antibodies, in contrast, are antibodies derived from non-human germline immunoglobulin sequences, but which have been modified such that the non-human sequences are replaced by human sequences. Humanized antibodies can be derived from the germline immunoglobulin sequences of, for example, a mouse, rat, rabbit, etc. Indeed, humanized antibodies can be derived from the germline immunoglobulin sequences of any other animal. As defined herein, antibodies are those derived from the V H Domain and V L If at least one of the domains is humanized, it is considered a humanized antibody. In particular, a humanized antibody can have a humanized V H Sequence and humanized V L It may contain sequences.

[0104] In humanized variable domains, non-human variable domain sequences are modified such that non-human (e.g., murine) framework sequences are replaced with human framework sequences; therefore, typically, the only non-human sequences in an antibody are CDR sequences. Antibody humanization is generally performed by a process known as CDR grafting, although any other technique in the art may be used. CDR grafting is fully described in Williams, DG et al., Antibody Engineering, Vol. 1, R. Kontermann and S. Dubel (eds.), Chapter 21, pp. 319-339. In this process, humanization of a non-human variable domain involves inserting the non-human CDRs from each immunoglobulin chain into the FRs of the most appropriate human variable region. This is done by aligning the non-human variable domain against a database of known human variable domains (e.g., IMGT or Kabat). Suitable human framework regions are identified from the most well-aligned variable domains, for example, those with high sequence identity between the human and non-human framework regions, those containing CDRs of the same length, those with the most similar structure (based on homology modeling), etc. Non-human CDR sequences are then grafted into the appropriate positions of the lead human framework sequence using recombinant DNA technology, and humanized antibodies are then produced and tested for binding to the target antigen. The process of antibody humanization is known and understood by those skilled in the art, and those skilled in the art can carry out the techniques without further instruction. Antibody humanization services are also provided by several commercial companies, such as GenScript (USA / China) or MRC Technology (UK). Humanized antibody fragments can be easily obtained from humanized antibodies as described above.

[0105] Alternatively, chimeric antibodies, rather than human or humanized antibodies, may be used in the methods presented herein. Chimeric antibodies are antibodies having variable domains derived from one species and constant domains derived from another species. Thus, for example, a chimeric antibody for use herein may contain a non-human variable domain (e.g., a mouse-derived variable domain or a rat-derived variable domain) and a human constant domain. Chimeric antibodies can be produced using any suitable technique, for example, recombinant DNA technology, in which a DNA sequence for a variable domain (e.g., a non-human variable domain) is fused with a DNA sequence for a constant domain (e.g., a human constant domain) to encode a chimeric antibody. Chimeric antibody fragments can be obtained either by using recombinant DNA technology to generate a DNA sequence encoding such a polypeptide, or by processing a chimeric antibody to generate the desired fragment, as described above.

[0106] As used herein, an antibody or fragment thereof may be an isolated antibody or an isolated antigen-binding fragment of an antibody. An "isolated antibody" or "isolated antigen-binding fragment of an antibody," as used herein, refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody or antigen-binding fragment that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" or "isolated antigen-binding fragment of an antibody" for purposes of the methods provided herein. An isolated antibody or fragment thereof also encompasses an antibody or fragment thereof in situ within a recombinant cell. An isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has been subjected to at least one purification or isolation step. An isolated antibody or antigen-binding fragment thereof may be substantially free of other cellular material and / or chemicals.

[0107] Thus, antibodies or fragments thereof for use herein can be synthesized by any method known in the art. In particular, antibodies or fragments thereof can be synthesized using a protein expression system, for example, a cellular expression system using a prokaryotic (e.g., bacterial) host cell or a eukaryotic (e.g., yeast, fungal, insect, or mammalian) host cell. Cells that can be used to produce antibodies or fragments thereof are further discussed below. An alternative protein expression system is a cell-free in vitro expression system in which a nucleotide sequence encoding a specific binding molecule is transcribed into mRNA in vitro, and the mRNA is translated into protein. Cell-free expression system kits are widely available and can be purchased, for example, from ThermoFisher Scientific (USA). Alternatively, antibodies and fragments thereof can be chemically synthesized in a non-biological system. Liquid-phase or solid-phase synthesis can be used to generate polypeptides that can form or be contained within the antibodies or fragments thereof used herein. Those skilled in the art can easily produce antibodies or fragments thereof using appropriate methodologies common in the art. In particular, antibodies or fragments thereof can be recombinantly expressed in mammalian cells such as CHO cells. Other suitable mammalian cells for producing antibodies or fragments thereof for use herein include monkey kidney cells (e.g., COS-7), HEK293 HeLa cells, baby hamster kidney (BHK) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and several other cell lines, including the mouse myeloma cell lines NSO and SP2 / 0.

[0108] When cultured under appropriate conditions, host cells synthesize antibodies or antigen-binding fragments thereof for use herein. The antibodies or antigen-binding fragments thereof can then be collected from the culture medium (if the host cells secrete the antibodies or antigen-binding fragments into the medium) or directly from the host cells producing them (if the antibodies or antigen-binding fragments are not secreted). Thus, antibodies or fragments thereof can be isolated from synthesis, as described above. The selection of an appropriate host cell depends on various factors, such as the desired expression level, polypeptide modifications (e.g., glycosylation or phosphorylation) desired or necessary for activity, and the ease of folding into a biologically active molecule. Host cell lines producing antibodies or fragments thereof for use herein may stably express the antibodies or fragments thereof, or may transiently express the antibodies or fragments thereof.

[0109] The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as the paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. An epitope may be a conformational epitope or a linear epitope. A conformational epitope arises from the spatial arrangement of amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope that arises from adjacent amino acid residues within a polypeptide chain. In certain circumstances, an epitope may include a sugar, phosphoryl, or sulfonyl moiety on an antigen.

[0110] Antibodies for use in the methods provided herein may bind to any suitable epitope of PAR2; i.e., the antibody may bind to any epitope, so long as binding to that epitope has the desired effect of inhibiting PAR2 activity. In certain embodiments, antibodies or fragments thereof for use in the methods bind to PAR2 at an epitope comprising the amino acid sequence set forth in SEQ ID NO: 11. That is, antibodies or fragments thereof for use herein may specifically bind to the amino acid sequence set forth in SEQ ID NO: 11. SEQ ID NO: 11 corresponds to amino acids 33-48 of full-length PAR2. This sequence contains the PAR2 cleavage site (located between amino acids 4 and 5 of SEQ ID NO: 11), and therefore, antibody binding to this epitope inhibits PAR2 cleavage and activation. SEQ ID NO: 11 is the epitope of MEDI0618 determined by X-ray crystallography of the complex between MEDI0618 Fab and the N-terminal PAR2 polypeptide (not shown in the Examples).

[0111] Whether an antibody against PAR2 binds to PAR2 at the epitope of SEQ ID NO: 11 can be determined by epitope mapping. Any method for epitope mapping known in the art can be used for this purpose. Examples of such methods include HDX, epitope excision, peptide panning, X-ray cocrystallography, NMR, etc. (Clementi et al., Methods Mol. Biol. 1131:427-446, 2014; Abbott et al., Immunology 142(4):526-535, 2014). An antibody or fragment thereof that specifically binds to SEQ ID NO: 11 can also be specifically generated using the peptide of SEQ ID NO: 11 as an antigen, for example, by immunizing an animal (e.g., a mouse) with the peptide of SEQ ID NO: 11, or by using the peptide of SEQ ID NO: 11 as an affinity partner when generating antibodies by phage display.

[0112] For the avoidance of doubt, an anti-PAR2 antibody or fragment thereof may be considered to recognize (i.e., specifically bind) the epitope of SEQ ID NO: 11 if the antibody or fragment thereof binds to the native sequence of PAR2 (SEQ ID NO: 14) at the sequence shown in SEQ ID NO: 11. If the sequence of SEQ ID NO: 11 is mutated in PAR2, an antibody that specifically binds to SEQ ID NO: 11 may nevertheless bind to the mutated sequence, i.e., mutations in the epitope sequence of SEQ ID NO: 11 may be tolerated. However, an antibody that specifically binds to the epitope of SEQ ID NO: 11 will not bind to any other epitope on PAR2, or at least will recognize the epitope of SEQ ID NO: 11 more strongly than any other epitope on PAR2.

[0113] An antibody or fragment thereof can be considered to bind PAR2 at SEQ ID NO: 11 if it binds to PAR2 at an epitope sequence within SEQ ID NO: 11, even if the antibody or fragment does not bind to the entire peptide of SEQ ID NO: 11. Similarly, an antibody or fragment thereof can be considered to bind PAR2 at SEQ ID NO: 11 if the epitope for the antibody or fragment includes SEQ ID NO: 11 (or a portion of SEQ ID NO: 11), but is not contained exclusively within SEQ ID NO: 11. Thus, an antibody or fragment thereof can be considered to bind SEQ ID NO: 11 if the antibody or fragment binds to an epitope that consists in part of SEQ ID NO: 11 (or a portion of SEQ ID NO: 11) and in part of another sequence, for example, a sequence immediately N-terminal or C-terminal to SEQ ID NO: 11 within PAR2.

[0114] Thus, the antibody or fragment thereof may bind to an epitope comprising SEQ ID NO:11, or an epitope comprising a part of SEQ ID NO:11, or an epitope consisting of SEQ ID NO:11, or an epitope consisting of a part of SEQ ID NO:11.

[0115] As described above, it may be advantageous for the antibodies used in the methods provided herein to bind to PAR2 with higher affinity at physiological, extracellular pH (approximately pH 7.4) than at slightly acidic pHs, such as approximately pH 6.0. Anti-PAR2 antibodies with such binding characteristics have been found to exhibit enhanced biological efficacy compared to anti-PAR2 antibodies whose binding to PAR2 is not affected by pH. Without being bound by theory, this effect is believed to be due to the pH-dependent target binding, which allows the antibody to be recycled after binding to PAR2. The extracellular domain of PAR2 is located outside the cell, where the pH is approximately 7.4. When an anti-PAR2 antibody binds to PAR2, PAR2 is internalized, where it enters endosomal and lysosomal intracellular compartments. Both endosomal and lysosomal intracellular compartments are acidic (endosomes have a pH in the range of approximately 5.5 to 6). If the antibody binds to PAR2 in a pH-dependent manner as described above, the antibody dissociates from PAR2 upon internalization of the antibody-PAR2 complex into the endosome, due to a decrease in the endosomal pH. This allows the antibody to be recycled in the endocytosis cycle and released extracellularly again rather than being degraded, thereby increasing the serum half-life of the antibody and therefore improving its pharmacokinetic profile in therapeutic settings.

[0116] Antibodies may exhibit pH-dependent recognition of PAR2 if they contain at least one histidine residue in the CDR. a is approximately 6.0, and therefore, below this pH level, the imidazole ring of the histidine side chain is generally protonated (and therefore neutral), whereas above this pH level, the imidazole ring is generally unprotonated (and therefore positively charged). This change in the charge of the histidine side chain can substantially alter the affinity for its target of an antibody containing one or more histidine residues in its CDRs.

[0117] The antibodies or fragments thereof for use herein may contain histidine in VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and / or VHCDR3. The antibodies or fragments thereof for use herein may contain a total of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more histidine residues in the CDR sequences. Such histidine residues may be naturally present in the CDR sequences or may be introduced by mutation (e.g., site-directed mutagenesis). The histidine residues may be present at any position in the CDR sequences.

[0118] Antibodies for use herein may bind to PAR2 with the above-mentioned affinities at pH 7.4 and 6.0. For example, antibodies for use herein may bind to PAR2 with an affinity at pH 7.4 that is at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater at pH 7.4 than at pH 6.0.

[0119] Antibodies or antigen-binding fragments thereof for use in the methods provided herein particularly have the following CDRs: VLCDR1 comprising the sequence shown in SEQ ID NO: 1; VLCDR2 comprising the sequence shown in SEQ ID NO:2; VLCDR3 comprising the sequence shown in SEQ ID NO:3; VHCDR1 comprising the sequence shown in SEQ ID NO:4; VHCDR2 comprising the sequence set forth in SEQ ID NO:5; and VHCDR3 comprising the sequence shown in SEQ ID NO:6 may include: As described above, the CDRs can consist of SEQ ID NOs: 1 to 6, respectively.

[0120] An antibody or antigen-binding fragment thereof for use herein comprises a heavy chain variable domain (V) comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:7. H ).

[0121] The antibody or antigen-binding fragment thereof for use herein comprises a light chain variable domain (V) comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 8. L ).

[0122] In particular, the antibodies or antigen-binding fragments thereof for use herein are (i) a heavy chain variable domain (V) comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 7; H ); and (ii) a light chain variable domain (V) comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 8; L ) may include:

[0123] The antibody or antigen-binding fragment thereof has at least 70% identity to SEQ ID NO:7, but less than 100% identity to SEQ ID NO:7. H , this may be subject to the proviso that the CDRs are as defined above, i.e. VHCDR1 comprises the amino acid sequence of SEQ ID NO: 4, VHCDR2 comprises the amino acid sequence of SEQ ID NO: 5 and VHCDR3 comprises the amino acid sequence of SEQ ID NO: 6. Thus, if a heavy chain variable region comprises a variant of SEQ ID NO: 7, generally all of the variation in the heavy chain variable domain sequence compared to SEQ ID NO: 7 will be found within the framework regions.

[0124] Similarly, an antibody or antigen-binding fragment thereof may have a V that is at least 70% identical to SEQ ID NO:8, but less than 100% identical to SEQ ID NO:8. LVLCDR1 comprises the amino acid sequence of SEQ ID NO: 1, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 2 and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 3. Thus, if a light chain variable domain comprises a variant of SEQ ID NO: 8, generally all of the variation in the light chain variable domain sequence compared to SEQ ID NO: 8 will be found within the framework regions.

[0125] An antibody or antigen-binding fragment thereof for use herein may comprise a heavy chain comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:9.

[0126] An antibody or antigen-binding fragment thereof for use herein may comprise a light chain comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO:10 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:10.

[0127] In particular, antibodies for use herein are (i) a heavy chain comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO: 9; and (ii) a light chain comprising (or consisting of) the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 10 may include:

[0128] As mentioned above, if an antibody comprises a heavy chain that is a variant of SEQ ID NO: 9, this may be subject to the proviso that the CDRs are as defined above, i.e. VHCDR1 comprises the amino acid sequence of SEQ ID NO: 4, VHCDR2 comprises the amino acid sequence of SEQ ID NO: 5 and VHCDR3 comprises the amino acid sequence of SEQ ID NO: 6. That is, if the heavy chain comprises a variant of SEQ ID NO: 9, generally all of the variation in the heavy chain sequence compared to SEQ ID NO: 9 will be found within the framework regions of the constant and variable domains.

[0129] Similarly, if an antibody comprises a light chain that is a variant of SEQ ID NO: 10, this may be subject to the proviso that the CDRs are as defined above, i.e. VLCDR1 comprises the amino acid sequence of SEQ ID NO: 1, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 2 and VLCDR3 comprises the amino acid sequence of SEQ ID NO: 3. That is, if the light chain comprises a variant of SEQ ID NO: 10, generally all of the variations in the light chain sequence compared to SEQ ID NO: 10 will be found within the framework regions of the constant and variable domains.

[0130] The antibody having the heavy chain amino acid sequence set forth in SEQ ID NO: 9 and the light chain amino acid sequence set forth in SEQ ID NO: 10 is referred to herein as MEDI0618.

[0131] The sequence identity of the above-mentioned sequence variants can be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform pairwise or multiple alignment of sequences are useful, for example, EMBOSS Needle or EMBOSS stretcher (both Rice, P. et al., Trends Genet. 16, (6) 276-277, 2000) can be used for pairwise sequence alignment, Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. 32(5):1792-1797, 2004) can be used for multiple sequence alignment, or any other suitable program can be used. Regardless of whether the alignment is pairwise or multiple, it must be performed globally (i.e., across the entire reference sequence) rather than locally.

[0132] Sequence alignments and percent identity calculations may be determined, for example, using standard Clustal Omega parameters: Gonnet matrix, gap opening penalty of 6, gap extension penalty of 1. Alternatively, standard EMBOSS Needle parameters may be used: BLOSUM62 matrix, gap opening penalty of 10, gap extension penalty of 0.5. Alternatively, any other suitable parameters may be used.

[0133] For the purposes of this application, in case of discrepancies between sequence identity values ​​obtained by different methods, the value obtained by global pairwise alignment using EMBOSS Needle with default parameters shall be considered valid.

[0134] Variants of the sequences shown herein (i.e., sequences having at least 70% sequence identity to SEQ ID NO: 7, 8, 9 or 10) may be obtained by substitution, deletion or insertion of amino acid residues compared to the original sequences.

[0135] When a sequence is altered by the substitution of a specific amino acid residue, the substitution may be a conservative amino acid substitution. As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Amino acids with similar side chains tend to have similar properties, and therefore, conservative substitution of an amino acid that is important for the structure or function of a polypeptide may be expected to have a smaller impact on the polypeptide structure / function than a non-conservative amino acid substitution at the same position. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, conservative amino acid substitutions can be considered as substitutions in which a specific amino acid residue is replaced with a different amino acid from the same family. However, substitutions of amino acid residues can also be considered as non-conservative substitutions in which one amino acid is replaced with another amino acid having a side chain from a different family.

[0136] When antibodies or fragments thereof are used that contain variants of MEDI0618 variable domain sequences (i.e., variable domains that are variants of SEQ ID NO:7 or SEQ ID NO:8, as described above) or complete chain sequences (i.e., heavy or light chains that are variants of SEQ ID NO:9 or SEQ ID NO:10), the variants may have equivalent activity to MEDI0618 or the corresponding fragments thereof. For example, antibodies that are variants of MEDI0618 (i.e., antibodies comprising the variant sequences described above) may bind to PAR2 with an affinity equivalent to MEDI0618 that is no less than, or no less than substantially less than, the affinity with which MEDI0618 binds to PAR2. For example, MEDI0618 variants may bind to PAR2 with an affinity equivalent to that of MEDI0618 that is no less than, or no less than, the affinity with which MEDI0618 binds to PAR2. D Alternatively, a variant of MEDI0618 may be considered to bind PAR2 with an affinity that is not substantially less than the affinity with which MEDI0618 binds to PAR2 if the variant of MEDI0618 binds to PAR2 with an affinity that is reduced by no more than 5%, no more than 10%, no more than 15%, no more than 20%, or no more than 25% compared to the affinity of MEDI0618.

[0137] Antibodies that are variants of MEDI0618 may exhibit other biological activities equivalent to those exhibited by MEDI0618, such as PAR2 inhibition (measured by calcium flux assays as described above), PAR2 internalization, and any other effector function exhibited by MEDI0618.

[0138] The anti-PAR2 antibodies used in the methods provided herein can be IgG antibodies that contain a constant domain (Fc domain) that contains one or more mutations, relative to the native Fc sequence, that enhance or decrease antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the anti-PAR2 antibodies used herein can be IgG antibodies that contain one or more mutations, relative to the native Fc sequence, that enhance or decrease antibody binding to the FcRn receptor at, for example, acidic pH compared to neutral pH. H Area 2 or C HThe antibody may contain mutations in the three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include a modification at position 250 (e.g., E or Q); a modification at position 252 (e.g., L, Y, F, W, or T); a modification at position 254 (e.g., S or T); a modification at position 256 (e.g., S, R, Q, E, D, or T); a modification at position 259 (e.g., I); a modification at position 265 (e.g., A); a modification at position 297 (e.g., A); a modification at position 307; a modification at position 308 (e.g., F or P); a modification at position 428 (e.g., L or F); a modification at position 433 (e.g., H, L, R, S, P, Q, or K); and a modification at position 434 (e.g., H, S, F, or Y). In these exemplary modifications, the position number refers to the amino acid position in a full-length IgG heavy chain (i.e., position 1 is defined as the first amino acid in the variable domain, excluding the signal sequence). In particular, the position number refers to the amino acid position in a full-length human IgG heavy chain, particularly a full-length human IgG1 heavy chain. The amino acids listed are residues that may be present in the modified chain at the listed position (ie, amino acids that may be substituted for the native amino acid).

[0139] Particular combinations of alterations that the Fc domain may contain include alterations at positions 250 and 428; 252, 254 and 256; 307, 308 and / or 434; and 428, 433 and / or 434 (as described above). For example, an Fc domain can include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I) and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and a 428L modification (e.g., T250Q and M428L); a 307 and / or a 308 modification (e.g., 308F or 308P); and / or a 265 (e.g., 265A, e.g., D265A) and / or a 297 (e.g., 297A, e.g., D297A) modification. Indeed, an Fc domain can include any combination of the above modifications.

[0140] The antibody may contain the triple mutation L234F / L235E / P331S ("TM") in the Fc domain. TM causes a significant reduction in the binding activity of human IgG1 molecules to human Fcγ receptors, including FcγR1 (CD64), FcγR2A (CD32A), and CD16 (FcγRIII), as detailed, for example, in Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 64:700-704 (2008). Antibodies with increased half-lives can also be generated by modifying amino acid residues identified to be involved in the interaction between the Fc and FcRn receptors. For example, the triple mutation M252Y / S254T / T256E ("YTE") can be inserted into the C region of a human IgG molecule, as described in U.S. Pat. No. 7,083,784, the entire contents of which are incorporated herein by reference. H The introduction of the YTE modification into the 2 domain results in increased binding of these human IgG molecules to the human fetal Fc receptor (FcRn). The antibodies used herein may contain the YTE modification.

[0141] The antibody or antibody fragment thereof used in the present method can be an antibody or antibody fragment that has a different sequence from MEDI0618 (e.g., has different CDRs from MEDI0618 or has different variable domain framework region sequences) but is bioequivalent to MEDI0618 (or is bioequivalent to the corresponding fragment of MEDI0618). Two antibodies or antigen-binding fragments thereof are considered to be bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not demonstrate significant differences in the rate and extent of absorption when administered under similar experimental conditions at the same molar dose, either in single or multiple doses. Some antibodies or antigen-binding fragments are considered equivalents or pharmaceutical substitutes if they have an equivalent degree of absorption but not an equivalent rate of absorption, but are nevertheless considered bioequivalent because such differences in absorption rate are intentional, labeled, not essential, for example, to achieving effective body drug concentrations during long-term use, and are not considered medically significant with respect to the particular drug product being tested.

[0142] Two antibodies or antigen-binding fragments thereof may be considered bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. Alternatively or additionally, two antibodies or antigen-binding fragments thereof may be considered bioequivalent if a patient can switch between the two products one or more times without the expected risk of adverse effects, including clinically significant changes in immunogenicity, or reduced efficacy, compared to continuing therapy without such a switch. Alternatively or additionally, two antibodies or antigen-binding fragments thereof may be considered bioequivalent if they both act via a common mechanism of action for the condition for which they are intended, to the extent such mechanism is known. For example, an antibody that is bioequivalent to MEDI0618 may bind to the same epitope (i.e., SEQ ID NO: 11) as MEDI0618 with similar affinity, thereby causing the same degree of inhibition of PAR2 activity as MEDI0618 (e.g., as measured by the calcium flux assay described above).

[0143] Bioequivalence may be demonstrated by in vivo and in vitro methods. Criteria for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data; (c) in vivo studies in humans or other mammals that measure the appropriate acute pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0144] Therapeutic Formulations and Administration The antibody or antigen-binding fragment thereof may be administered to a subject in a pharmaceutical composition containing at least one pharmaceutically acceptable diluent, carrier, or excipient. As used herein, "pharmaceutically acceptable" refers to a component that is compatible with the other components of the composition and physiologically acceptable to the recipient. The nature, dosage, and the like of the composition and carrier or excipient materials may be selected in a routine manner, depending on the selected and desired route of administration, etc. The dosage may likewise be determined in a routine manner and may depend on the nature of the molecule, the age of the patient, the mode of administration, etc.

[0145] Pharmaceutical compositions for use herein include liquid solutions or syrups, solid compositions such as powders, granules, tablets, or capsules, creams, ointments, and any other form commonly used in the art. Pharmaceutically acceptable diluents, carriers, and excipients suitable for use in such compositions are well known in the art. For example, suitable excipients include lactose, corn starch or its derivatives, stearic acid or its salts, vegetable oils, waxes, fats, and polyols. Suitable carriers or diluents include carboxymethylcellulose (CMC), methylcellulose, hydroxypropylmethylcellulose (HPMC), dextrose, trehalose, liposomes, polyvinyl alcohol, pharmaceutical-grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, gelatin, oils, alcohols, surfactants, and emulsifiers, such as polysorbates. Stabilizing agents, wetting agents, emulsifying agents, sweeteners, and the like may also be used.

[0146] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: a sterile diluent, such as water for injection, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as a solvent or suspending medium; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers, such as acetic acid, citric acid, or phosphate, and agents for adjusting tonicity, such as dextrose. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0147] The mode of administration of the antibody or antigen-binding fragment thereof for use herein can be selected by a skilled physician. Similarly, the appropriate dosage can be determined by one skilled in the art, taking into account the age and size of the patient, the route of administration, etc. The preferred dose is typically calculated according to body weight or body surface area. The frequency and duration of treatment can be adjusted depending on the severity of the condition. The effective dosage and administration schedule of the anti-PAR2 antibody or antigen-binding fragment thereof can be determined empirically. For example, the patient's progress can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. The increase or decrease in dosage between species can be performed by methods well known in the art (see, for example, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0148] Other Aspects As mentioned above, provided herein are methods for treating migraine in a subject using a PAR2 antibody. Alternatively, these methods include: (i) an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject who is an inadequate responder to anti-CGRP therapy (aCGRP-IR) or who suffers from migraine that does not respond to anti-CGRP therapy or an inhibitor of calcitonin gene-related peptide (CGRP); (ii) an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject suffering from migraine and who is intolerant to anti-CGRP therapy or an inhibitor of calcitonin gene-related peptide (CGRP); (iii) use of an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 in the manufacture of a medicament for treating migraine in a subject who is an inadequate responder to anti-CGRP therapy (aCGRP-IR) or who is unresponsive to anti-CGRP therapy or calcitonin gene-related peptide (CGRP) inhibitor therapy; and (iv) The present invention may be considered to provide use of an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 in the manufacture of a medicament for treating migraine in a subject suffering from migraine and who is intolerant to anti-CGRP therapy.

[0149] All aspects of the use of the antibodies (and fragments thereof) of (i) and (ii) and the antibodies (and fragments thereof) of (iii) and (iv) for treating migraine may be as defined above with respect to the methods presented herein.

[0150] All documents cited herein are hereby incorporated by reference in their entirety.

[0151] The invention may be further understood by reference to the drawings and the following non-limiting examples. [Example]

[0152] Materials and Methods Assessment of periorbital and hindpaw response frequency Periorbital and hind paw response frequencies to tactile stimulation were measured before and after the epidural injection. Mice were placed in a Plexiglas chamber (L x W x H = 4 in x 4 in x 8 in) and secured with a wire mesh platform (0.635 cm 2The rats were placed on a grid (with a 30° angle) for 2 hours. After habituation, 0.4 g and 1 g von Frey filaments (Stoelting, Wood Dale, IL, USA) were applied 10 times to the periorbital and hind paw regions, respectively. The filaments were gently applied until they were slightly arched. For the periorbital region, a positive response characterized by facial grooming, head shaking, and / or turning away the head after filament application was scored after each filament application. For the hind paw region, a positive response was characterized by rapid paw withdrawal, paw shaking, and / or paw licking. Any such behaviors exhibited before filament application were not considered positive responses. Migraine-like pain behavior, observed by an increase in response frequency, was considered to reflect the development of cutaneous allodynia (CA). Response frequency was calculated by [(number of positive responses × 100) / 10].

[0153] epidural injection Under mild and brief isoflurane anesthesia, the epidural injection procedure, which allows minimally invasive administration of substances onto the dura mater of mice through the junction of the sagittal and lambdoid sutures, was performed as previously described by Burgos-Vega and colleagues (Cephalalgia 39:123–134, 2019) with minor modifications. The epidural syringe was modified from a commercially available cannula (Plastic One / lnvivo1, part #C313I / SPC, Internal Cannula, Standard, 28 gauge). The syringe's protrusion length was adjusted to 0.7 mm and controlled by a stopcock to limit its length and maintain the integrity of the dura mater. The syringe was connected to Tygon tubing (Cole Parmer Co., Vernon Hills, Illinois, USA) attached to a 25 mL Hamilton syringe (Hamilton, Reno, Nevada, USA). The syringe was inserted through the junction of the sagittal and lambdoid sutures and the injection was delivered onto the dura mater. The total injection volume was 5 mL, and each syringe was used a maximum of four times.

[0154] Individual in vivo experimental designs Experimental Design 1 Effect of MEDI0618 on periorbital and hindpaw allodynia induced by epidural administration of inflammatory mediators (IM) in female C57BL6J mice.

[0155] Four animals per group received 50 mg / kg MEDI0618 IgG1™ or 50 mg / kg isotype control (NIP228 IgG1™; concentration 10.7 mg / ml) subcutaneously. All mice received inflammatory mediators (IM) intrathecally.

[0156] Protocol: Mice were acclimated to von Frey (VF) for 3 days. A baseline 1 (BL1) measurement was performed, followed by antibody treatment 24 hours before the IM time course. During the IM time course, mice were acclimated to VF for 2 hours, followed by BL2 measurements. IM was administered epidurally, and VF measurements were taken 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after epidural IM administration. The IM mix contained bradykinin (1 mM), histamine (1 mM), 5-hydroxytryptamine (5-HT) (1 mM), and prostaglandin E2 (PGE2) (100 mM) in synthetic interstitial fluid (SIF), pH 5.0.

[0157] Experimental Design 2 Effect of pretreatment with 1 mg / kg intraperitoneal olcegepant (30 min before CGRP administration) on periorbital and hindpaw allodynia induced by epidural injection of CGRP in female mice.

[0158] Protocol: Mice were acclimated to VF for 2 hours each day over three days. Six animals per group were shaved 1 day before intraperitoneal treatment with olcegepant at time course / BL1 / 1 mg / kg / 10 mL (i.e., a dose of 1 mg / kg olcegepant in a volume of 10 mL / kg) or vehicle (20% DMSO / 80% saline). BL2 measurements were taken 15 minutes after the start of treatment, after which mice were injected epidurally with CGRP. Each mouse received 1 pg of CGRP in a volume of 5 μL using a 0.7 mm syringe. VF assessments were performed 5, 10, 20, 40, 60, 120, and 180 minutes after injection. Shaving and epidural administration were performed under light anesthesia with isoflurane.

[0159] Experimental Design 3 Effect of pretreatment with 1 mg / kg intraperitoneal olcegepant (30 min before CGRP) on periorbital and hindpaw allodynia induced by 5 μL IM epidural injection in female mice.

[0160] Protocol: Mice were acclimated to VF for 2 hours each over three days. There were six animals per group. Animal heads were shaved one day before intraperitoneal treatment with Time Course / BL1 / 1 mg / kg / 10 mL olcegepant or vehicle (20% DMSO / 80% saline). BL2 measurements were performed 15 minutes after the start of treatment, after which all mice were injected epidurally with 5 μL of IM (as defined above) using a 0.7 mm syringe. VF assessments were performed 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after injection. Shaving and epidural administration were performed under light anesthesia with isoflurane.

[0161] Experimental Design 4 Effect of MEDI0618 on periorbital and hindpaw allodynia induced by epidural administration of inflammatory mediators (IM) in female C57BL6J mice.

[0162] Four animals received 50 mg / kg MEDI0618 IgG1™ (Lot SP16-112) subcutaneously and four animals received 50 mg / kg isotype control (NIP228 IgG1™, Lot SP14-302; concentration 10.7 mg / ml) subcutaneously as in Experiment 1. All mice were administered IM intrathecally.

[0163] Protocol: Mice were acclimated to VF for 3 days. Animals' heads were shaved on day 3, followed by BL1 measurements, and then treated with MEDI0618 24 hours before the IM time course. During the IM time course, mice were acclimated to VF for 2 hours, followed by BL2 measurements. IM was then administered epidurally, and VF measurements were taken 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after epidural IM administration.

[0164] statistical analysis Statistical analysis for these studies was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA). For the analysis of time-course experiments to compare sensory thresholds between two or more groups, a two-way analysis of variance (ANOVA) followed by a Sidak or Tukey test was used, respectively. Flow cytometry quantification and area under the curve (AOC) were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey test. All data were expressed as mean-standard error of the mean (SEM), and statistical significance was set at p<0.05.

[0165] Mouse trigeminal nerve cultures Trigeminal neurons were collected from 6-8 week-old female C57BL6 mice as previously described (Malin et al., 2007 Nature Methods, DOI:10.1038 / nprot.2006.461). Briefly, after euthanasia and decapitation, the overlying skin, skull, and brain were removed to expose the trigeminal ganglion at the base of the skull. The trigeminal ganglion was dissected, minced, and subjected to sequential digestion with papain, followed by collagenase type II and dispase type II. Neurons were separated from myelin and neural debris by centrifugation through a Percoll gradient (12.5% ​​Percoll over 28% Percoll). Cells were diluted to high density by adding 30 μl of medium (L15 + 5% fetal bovine serum (FBS) + 2% 1 M HEPES + 1% penicillin / streptomycin) per trigeminal pair. A 40 μl volume of the cell mix was dropped onto a 12 mm coverslip pre-coated with poly-D-lysine (PDL) and laminin (Biocoat #1 German Glass) in a 24-well plate. After allowing cells to attach for 30 minutes, the wells were maintained with sufficient F12 (Ham) Nutrient Mix + 10% FBS + 1% penicillin / streptomycin. Trigeminal neuron cultures were incubated in vitro at 37°C and 5% CO2 for 2–4 days before calcium imaging experiments.

[0166] Trigeminal neuron calcium imaging Experiments were performed to record cytosolic calcium concentrations to measure CGRP receptor functional activity and PAR2 activation after treatment with specific agonists: rat CGRP peptide (CGRP; Tocris) and 2-furoyl-LIGRLO-amide (LIGRLO; Peptides International), respectively. Neuronal activity was distinguished from non-neuronal activity via responsiveness to 20 mM KCl. Mouse trigeminal cultures were loaded with 5 μM Fura-2 ratiometric calcium dye in Hank's balanced salt solution (HBSS) at 37°C and 5% CO2 for 30 min before being transferred to a perfusion chamber (Warner Instruments) containing HBSS. Wide-field calcium imaging was performed using an IX81 inverted microscope (Olympus) equipped with an sCMOS Orca R2 camera (Hamamatsu). Time series were recorded at a frame rate of 400 ms using sequential excitation at 340 nm (+ / - 25 nm) and 387 nm (+ / - 11 nm) and emission capture at 510 nm (+ / - 40 nm) bandpass. Cultures were perfused with HBSS + / - treatments at a flow rate of 1.1 ml per minute via a gravity-flow perfusion system equipped with a VC-6 valve controller (Warner Instruments) for switching treatment lines. After a baseline harvest in HBSS (80 s), cultures were treated sequentially with 1 μM CGRP (40 s), washout with HBSS (100 s), 10 μM LIGRLO (40 s), washout with HBSS (100 s), 20 mM KCI (30 s), and finally washout with HBSS (80 s). The order of CGRP and LIGRLO was routinely switched to eliminate data bias due to the order of treatments. Regions of interest (ROIs) were selected for putative neurons, and the ratio of fluorescence intensity (excitation at 387 nm to excitation at 340 nm) was calculated for each ROI for each time point. A custom-written Matlab routine was applied for trace quantification. First, traces with unstable baselines and traces belonging to non-neuronal cells were removed. Next, cells that were active during the treatment period with CGRP and LIGRLO were identified (ratio to baseline >10%).), and the maximum amplitude within these periods was calculated.

[0167] Calcium imaging in human dura mater cells Cell Culture: Human dural fibroblasts (Innoprot catalog number P10375) were used between P3 and P7. Primary human dural microvascular endothelial cells (Creative Biolabs; cat. number NCL-21P6-022) were used between P1 and P3. Cells were harvested from a 50% confluent T175 flask by treating with Accutase for 5 minutes, centrifuging at 1000 rpm for 5 minutes, and then resuspending in culture medium. Human dural fibroblast culture medium consisted of 500 ml of fibroblast basal medium; 10 ml of fetal bovine serum; 5 ml of 100x fibroblast growth supplement; and 5 ml of penicillin / streptomycin. Human dural microvascular endothelial cell culture medium consisted of Endothelial Growth Media 2 (EGM-2) plus commercially available supplements (Lonza). Cells were diluted to 333,333 cells per ml and plated at 5 × 10 cells per well in a Cell Coat Poly-D-Lysine-coated 384-well plate (black, opaque, #781946, Lot#E11020JH, Greiner). 3 The cells were then placed in a humidified incubator at 37°C for 24 hours before the start of the experiment.

[0168] To load the cells, the culture medium was removed from the 384-well plate. The cells were loaded with Fluo-8 (ScreenQuest™ Fluo-8 No Wash Calcium Assay Kit). The dye was added to HBSS (+Ca) according to the manufacturer's instructions. 2+ and Mg 2+ ) + 20 mM HEPES + 0.1% BSA (assay buffer) to a total volume of 10 ml per 384-well plate, and cells were loaded (20 μl / well) at 37°C for 30 minutes, followed by room temperature for 30 minutes.

[0169] Cells were assayed on a FLIPR Tetra (Molecular Devices) using an excitation wavelength of 470–495 nm and an emission wavelength of 515–575 nm at a frequency of 2 Hz. Drugs were prepared in assay buffer and added sequentially to the cell plate (10 μl per addition). To determine antibody (IgG1) potency, cells were pretreated with IgG diluted in assay buffer and allowed to stand at room temperature for up to 1 hour before the addition of 30 nM matriptase, a PAR2 agonist.

[0170] Analysis: The amplitude of the calcium response was determined by measuring the maximum-minimum (Max-Min) fluorescence from sweep 0 to the final sweep. Fluorescence data were plotted against the logarithm of the IgG concentration (M) and fitted as needed using "log(inhibitor) vs. response--Variable slope (four parameters)" in GraphPad Prism. Equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogEC50-X)) * Data are presented as mean + / - SEM with n=3. Results are representative of three independent experiments.

[0171] Pharmacokinetic study in rats A rat pharmacokinetic study was conducted in male Sprague-Dawley rats (200-215 g) at an intravenous dose level of 1 mg / kg. Monoclonal antibodies (ParB0048, ParB0103, ParB0120, ParB01288, ParB0129, ParB0139) were administered via tail vein dosing in a vehicle of 20 mM histidine / histidine HCl, 80 mM arginine, 120 mM sucrose, 0.02% polysorbate 80, pH 6.0, at a dose volume of 5 ml / kg, with n=3 rats per antibody dose group.

[0172] After intravenous administration, blood samples were collected over 14 days (0.5, 2, 6, 24, 48, 96, 168, 240, and 336 hours after dosing). At each time point, 150 μL of blood sample was collected from the tail vein and allowed to stand for a minimum of 30 minutes before centrifugation (13,000 g for 2 minutes), and the resulting serum was sampled. Quantification of monoclonal antibodies in each rat serum sample was performed using a universal assay format on a Gyrolab platform (Gyros). A Gyrolab Bioaffy 200 CD containing a pre-packed column of streptavidin-coated beads was functionalized with a biotinylated capture antibody (mouse anti-human IgG (CH2 domain; R10Z8E9)). Standards and samples were passed through the column, allowing the capture antibody to bind to the monoclonal antibody drug in the serum. A fluorophore-labeled detection antibody (sheep anti-human IgG H+L chain) was then passed through the column to bind to the captured analyte. The concentration of the detection reagent bound to the analyte (and therefore the concentration of the analyte) was calculated from the fluorescence measured by the fluorescence detector of the Gyrolab Workstation.

[0173] After analyte quantification, pharmacokinetic parameters were estimated using Phoenix WinNonlin pharmacokinetic software version 1.4 (Pharsight, USA) using a noncompartmental approach consistent with the intravenous infusion route of administration. All parameters were generated from individual serum concentrations and estimated using sample collection times (within acceptable limits) relative to the start of each dose administration. The area under the serum concentration-time curve (AUC) was calculated using the log-increasing, linearly decreasing trapezoidal method with linear interpolation. Where practical, the terminal elimination phase of each concentration-time curve was identified using at least the last three observed concentration values. The slope of the terminal elimination phase was determined using log-linear regression on unweighted concentration data. Parameters relying on the determination of the terminal elimination phase were excluded from summary statistics if three times the t estimate exceeded the sampling period, if the coefficient of determination was less than 0.800, and / or if the extrapolation of the AUC to infinity accounted for more than 30% of the total area. Clearance was determined as the ratio of dose and AUC to infinity.

[0174] decision Using a mouse model of migraine-like pain, we investigated the therapeutic effect of the anti-PAR2 antibody MEDI0618 for this indication. Migraine pain is known to be mediated by CGRP, and therefore administration of a CGRP inhibitor would be predicted to prevent the onset of migraine-like pain in the mouse model.

[0175] As expected, in a mouse model of migraine-like pain induced by epidural administration of CGRP, pretreatment with the CGRP inhibitor olcegepant effectively prevented the onset of migraine-like pain, as measured by the frequency of responses to tactile stimulation of von Frey hairs applied to the periorbital area (Figure 1B). These results confirmed that a 1 mg / kg dose of olcegepant was capable of completely inhibiting CGRP-induced pain and was therefore maximally effective. However, in a model of migraine-like pain induced by epidural administration of inflammatory mediators, pretreatment with the CGRP inhibitor olcegepant did not affect the onset of migraine-like pain (Figure 1A). This suggests the existence of a CGRP-independent pathway in migraine pain that is not affected by treatment with a CGRP inhibitor.

[0176] In the same model in which migraine-like pain was induced in mice by epidural administration of inflammatory mediators, pretreatment with the antagonist anti-PAR2 antibody MEDI0618 effectively prevented the onset of migraine-like pain, as measured by the frequency of responses to tactile stimulation of von Frey hairs applied to the periorbital region (Figures 2A-C) or hindpaw region (Figures 2D-E) (Figure 2). As shown in Figures 2C and 2F, administration of MEDI0618 reduced migraine-like pain behavior by more than 90%. This demonstrates that the antagonist anti-PAR2 antibody MEDI0618 is therapeutically effective against CGRP-independent migraine pain.

[0177] Single-cell calcium imaging experiments in mouse trigeminal neurons (Figure 3) show that the majority of functional responders to the PAR2 agonist LIGRLO (24% of trigeminal neurons) are not activated by CGRP. Only a small percentage (14%) of LIGRLO-activated neurons showed simultaneous activation of both LIGRLO and CGRP, suggesting that PAR2 and CGRP-R expression are distinct neuronal subpopulations. LIGRLO treatment exhibited higher amplitude calcium transient responses compared with CGRP. PAR2 expression in distinct neuronal subpopulations and the ability of PAR2 activation to directly affect CGRP-R expression and / or action potential firing may shape the differences in the PAR2 / CGRP pathway. The site of action of PAR2 in migraine may include non-neuronal cells of the trigeminovasculature, such as dural fibroblasts and dural microvascular endothelial cells. Activation of PAR2 by the endogenous serine protease matriptase generates calcium flux in both dural fibroblasts (Fig. 4A) and dural microvascular endothelial cells (Fig. 4B), which was completely and potently inhibited by MEDI0618 (IC in human dural fibroblasts and microvascular endothelial cells, respectively). 50 = 108 pM and 435 pM, respectively. The inhibitory effect of MEDI0618 on PAR2 was compared to that of the prior PAR2 antagonist antibody, PAR650097 (Kopruszinski et al., supra) by calcium flux assay. MEDI0618 demonstrated improved potency over PAR650097, as measured by its ability to inhibit matriptase-stimulated calcium influx in human dural fibroblasts (Figure 4A). MEDI0618 pC50 = 9.97 ± 0.26 M (n = 4); PAR650097 pC50 = 8.78 ± 0.15 M (n = 3), mean ± SD. Data shown represent exemplary data from one experiment.

[0178] A rat model was used to measure the serum clearance of MEDI0618 and several other related anti-PAR2 antibodies. As noted above, MEDI0618 was first described in WO2018 / 167322, where it is designated PaB670129. WO2018 / 167322 describes the generation of MEDI0618 from the parent antibody Par0067. In WO2018 / 167322, several variants of Par0067 were generated by mutating various residues within the VHCDR2, VHCDR3, and / or VLCDR3 of Par0067 to histidine to generate antibodies that exhibit pH-dependent binding. As noted above, pH-dependent binding is advantageous for therapeutic anti-PAR2 antibodies. As noted in the Sequence Listing, the MEDI0618 CDRs contain multiple histidine residues, all of which were introduced by mutation of the Par0067 CDR sequences. The serum clearance of MEDI0618 was compared in rats with that of the parent antibody Par0067 and a variety of other Par0067 derivatives containing histidine substitutions in the CDRs.

[0179] As shown in Figure 5, MEDI0618 (referred to as H129 in the figure legend) exhibits reduced serum clearance compared to the parent antibody (referred to as P67) and exhibits superior (i.e., slower) serum clearance (CL) than any of the other Par0067-derived antibodies tested, as also shown in the table below.

[0180] [Table 1]

[0181] Sequence Listing All sequences in this sequence listing are protein sequences.

[0182] [ka] TIFF2026504971000003.tif231168TIFF2026504971000004.tif73169

Claims

1. 1. A method for treating, alleviating, or preventing migraine in a subject, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of protease-activated receptor 2 (PAR2); The subject is an inadequate responder to anti-CGRP therapy (aCGRP-IR), or the subject suffers from migraine that is unresponsive to anti-CGRP (calcitonin gene-related peptide) therapy or to an inhibitor of calcitonin gene-related peptide.

2. 1. A method for treating, alleviating, or preventing migraine headache in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to and inhibits activity of protease-activated receptor 2 (PAR2); A method wherein the subject is intolerant to anti-CGRP (calcitonin gene-related peptide) therapy or an inhibitor of calcitonin gene-related peptide.

3. The method of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof specifically binds to human PAR2.

4. The method of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof prevents cleavage of the PAR2 extracellular domain.

5. The method of claim 4, wherein the antibody or antigen-binding fragment thereof prevents cleavage of PAR2 by trypsin, tryptase and / or matriptase.

6. The method of claim 5, wherein the antibody or antigen-binding fragment thereof interferes with the interaction of trypsin, tryptase and / or matriptase with PAR2.

7. The method of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof inhibits exposure of a PAR2-tethered ligand.

8. The method of any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof interferes with the interaction of a PAR2-tethered ligand with extracellular loop 2 of PAR2.

9. The method according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

10. The method of claim 9 , wherein the antibody is an IgG antibody.

11. Antigen-binding fragments of antibodies include Fab, Fab', F(ab') ２ The method according to any one of claims 1 to 8, wherein the antibody is an scFv or an scFv.

12. The method of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is a human antibody or a humanized antibody.

13. The method of any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof binds to PAR2 with higher affinity at pH 7.4 than at pH 6.

14. The antibody or antigen-binding fragment thereof (i) PAR2 has a K of less than about 5 nM, less than 1 nM, less than 900 pM, less than 800 pM, less than 700 pM, less than 650 pM, less than 600 pM, less than 500 pM, less than 200 pM, less than 100 pM, or less than 50 pM at pH 7.4 Ｄ and / or (ii) a K of PAR2 at pH 6 of greater than about 1 nM, greater than 5 nM, greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 40 nM, greater than 50 nM, greater than 60 nM, greater than 80 nM, or greater than 100 nM Ｄ Combine with The method of claim 13.

15. The method according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof specifically binds to the amino acid sequence shown in SEQ ID NO:

11.

16. The antibody or antigen-binding fragment thereof has the following CDRs: VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:1; VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4; VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6 The method according to any one of claims 1 to 15, comprising:

17. The antibody or antigen-binding fragment thereof (i) a V comprising the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 70%, 80%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; Ｈ domain; and (ii) V comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 70%, 80%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 Ｌ domain 17. The method of claim 16, comprising:

18. The antibody, (i) a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 70%, 80%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9; and (ii) a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 70%, 80%, or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

10.

18. The method of claim 17, comprising:

19. 19. The method of any one of claims 1 to 18, wherein the subject is an inadequate responder to migraine therapy with antibodies that prevent CGRP from binding to and / or activating the CGRP receptor, or the subject does not respond to migraine therapy with antibodies that prevent CGRP from binding to and / or activating the CGRP receptor or is intolerant to antibodies that prevent CGRP from binding to and / or activating the CGRP receptor.

20. 20. The method of claim 19, wherein the antibody that prevents CGRP from binding to and / or activating a CGRP receptor specifically binds to CGRP or a CGRP receptor.

21. 21. The method of any one of claims 1 to 20, wherein the subject is an inadequate responder to gepant-based migraine therapy, or is non-responsive to or intolerant to gepant-based migraine therapy.

22. 22. The method of any one of claims 1 to 21, wherein the subject has also failed to respond to migraine therapy with beta-blockers (e.g., propranolol), triptans (e.g., zolmitriptan), topiramate, candesartan, calcium channel blockers (e.g., flunarizine), tricyclic antidepressants (e.g., amitriptyline), and / or valproic acid or a salt thereof (e.g., sodium valproate).

23. 23. The method of any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof is administered in a pharmaceutical composition comprising at least one pharmaceutically acceptable diluent, carrier, or excipient.

24. The method of any one of claims 1 to 23, wherein the subject is a human.

25. 1. An antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject who is an inadequate responder to anti-CGRP therapy (aCGRP-IR) or a subject suffering from migraine who is unresponsive to anti-CGRP or unresponsive to therapy with an inhibitor of calcitonin gene-related peptide (CGRP).

26. 1. An antibody or antigen-binding fragment thereof that specifically binds to and inhibits the activity of human PAR2 for use in the treatment, alleviation, or prevention of migraine in a subject who is an inadequate responder to anti-CGRP therapy (aCGRP-IR) or in a subject suffering from migraine who is intolerant to anti-CGRP (calcitonin gene-related peptide) therapy or an inhibitor of calcitonin gene-related peptide (CGRP).

27. The antibody or antigen-binding fragment thereof for use according to claim 25 or 26, wherein the antibody, antigen-binding fragment thereof, subject, administration and / or therapeutic effect are as defined in any one of claims 3 to 24.

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