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WO2024156720A1 - Thérapie contre la migraine - Google Patents

Thérapie contre la migraine Download PDF

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Publication number
WO2024156720A1
WO2024156720A1 PCT/EP2024/051579 EP2024051579W WO2024156720A1 WO 2024156720 A1 WO2024156720 A1 WO 2024156720A1 EP 2024051579 W EP2024051579 W EP 2024051579W WO 2024156720 A1 WO2024156720 A1 WO 2024156720A1
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Prior art keywords
antibody
cgrp
par2
subject
antigen
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PCT/EP2024/051579
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English (en)
Inventor
John LINLEY
Iain Chessell
Frank Porreca
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MedImmune Ltd
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MedImmune Ltd
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Publication date
Application filed by MedImmune Ltd filed Critical MedImmune Ltd
Priority to CN202480014318.7A priority Critical patent/CN120835790A/zh
Priority to EP24702274.2A priority patent/EP4655068A1/fr
Priority to AU2024212070A priority patent/AU2024212070A1/en
Publication of WO2024156720A1 publication Critical patent/WO2024156720A1/fr
Priority to MX2025008572A priority patent/MX2025008572A/es
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin

Definitions

  • the method comprises administering to the patient an anti-PAR2 antagonistic antibody.
  • Migraine is a common neurological disorder primarily characterised by headache in the absence of injury, which may be severe and is often localised to one side of the head. Migraines are commonly recurring, in some individuals with high frequency. Recurrent migraines can be severely debilitating to sufferers, and with migraines experienced by around 20 % of adult women (and more than 5 % of adult men) according to the UK National Health Service, are a substantial economic and social burden.
  • migraine pain is ultimately caused by activation of nociceptors in the cranial meninges, which is believed to be caused by dural immune mast cell degranulation.
  • Mast cell degranulation results in the release of mediators including serine proteases such as tryptase and trypsin, which induce the release of neuropeptides from nerve endings, including the neuropeptide calcitonin gene-related peptide (CGRP) (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).
  • CGRP release is induced by serine proteases via the G-protein coupled receptor (GPCR) protease activated receptor 2 (PAR2).
  • GPCR G-protein coupled receptor
  • PAR2 protease activated receptor 2
  • the PAR family is known to include 4 members (PAR1-PAR4), activation of which has been found to be associated with inflammation and nociception (Gieseler et al., Cell Communication and Signalling 11 : 86, 2013).
  • PARs, including PAR2 are activated by proteolytic cleavage of their extracellular domains, which acts to unmask an N-terminal ‘tethered ligand’, i.e. a segment of the extracellular N-terminus of the PAR that, following cleavage, binds the activating sequences of the receptor.
  • PAR2 is activated by serine proteases including trypsin and tryptase (Gieseler et al., supra).
  • first line treatments include triptans, p-blockers (e.g. propranolol, topiramate (a carbonic anhydrase inhibitor) and candesartan (an angiotensin receptor blocker).
  • p-blockers e.g. propranolol, topiramate (a carbonic anhydrase inhibitor)
  • candesartan an angiotensin receptor blocker
  • a second line treatment may be prescribed such as a calcium channel blocker, a tricyclic antidepressant or valproic acid (or a salt thereof).
  • third line treatments have recently become available for patients for whom second line treatments are ineffective or inappropriate, which at present are primarily drugs which target the CGRP signalling axis.
  • third line drugs include gepants (CGRP receptor antagonists) and antibodies against CGRP and the CGRP receptor.
  • the present inventors have found that although PAR2 acts within the CGRP signalling pathway, PAR2 inhibition is effective in preventing both CGRP-dependent and CGRP-independent migraine pain (the pathway by which CGRP-independent migraine pain occurs remains to be elucidated).
  • a method for treating, alleviating or preventing migraine in a subject comprising administering to the subject an antibody or antigenbinding fragment thereof which specifically binds to protease activated receptor 2 (PAR2) and inhibits its activity, wherein the subject is an Inadequate Responder to anti-CGRP therapy (aCGRP-IR) or wherein the subject suffers from migraines which are not responsive to anti-CGRP therapy or to an inhibitor of calcitonin gene related peptide (CGRP).
  • PAR2 protease activated receptor 2
  • a method for treating, alleviating or preventing migraine in a subject in need thereof comprising administering to the subject an antibody or antigen-binding fragment thereof which specifically binds to protease activated receptor 2 (PAR2) and inhibits its activity, wherein the subject is intolerant to anti-CGRP (calcitonin gene-related peptide) therapy or to an inhibitor of calcitonin gene-related peptide (CGRP).
  • PAR2 protease activated receptor 2
  • CGRP calcitonin gene-related peptide
  • CGRP anti-CGRP
  • CGRP calcitonin gene-related peptide
  • CGRP calcitonin gene-related peptide
  • FIG. 1A-B show the effect of olcegepant (1 mg/kg i.p.) on migraine like pain behaviour induced by supradural administration of inflammatory mediators (IM) (A) or CGRP (B). Pain was indicated by the tactile frequency of response (%) in response to a von Frey hair applied to the periorbital region. Olcegepant was injected 30 mins prior to either IM or CGRP.
  • FIG. 2A-F show the effect of MEDI0618 on migraine pain-like behaviour induced by supradural administration of inflammatory mediators (IM) in female mice.
  • Data shows tactile frequency of response to a von Frey hair applied either to the periorbital or hindpaw region.
  • Antibody mAb, 50 mg/kg
  • BL1 Inflammatory mediators were injected supradurally after baseline 2 (BL2).
  • Statistical difference from control BL2 is indicated by ** (p ⁇ 0.01) *** (p ⁇ 0.001).
  • FIG. 3 shows that PAR2 is functionally expressed in a distinct population of TG neurons which do not necessarily co-express the CGRP receptor.
  • the results of single cell calcium imaging from adolescent mouse trigeminal ganglion neurons found 24% of neurons respond across 4 biological n with an increase in cytosolic calcium to a PAR2 agonist (2- Furoyl-LIGRLO-amide, 10 pM). Of these PAR2 agonist activated neurons only 14% were coactivated by CGRP (1 pM).
  • the left panel shows individual cell data with arrows indicating time at which agonists were applied.
  • Neuroon Stim indicates addition of a high potassium buffer (20 mM) to stimulate all neurons.
  • FIG. 4A-B shows that MEDI0618 demonstrates superior potency vs. PAR650097 (Kopruszinski et al., Cephalagia) and efficacy 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 addition of matriptase (30 nM).
  • Isotype control antibody NIP 229 h IgG 1 .
  • migraine migraine
  • migraine 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”.
  • the terms are used interchangeably herein.
  • the methods comprise administering to a subject an antibody that binds PAR2 and inhibits its activity, or an antigen-binding fragment thereof.
  • a “subject” as referred to herein may alternatively by referred to as a “patient”.
  • preventing migraine is meant herein that the method prevents a migraine developing after administration of the antibody (or fragment thereof) to the subject.
  • Preventing migraine includes complete prevention of migraines in the subject for a certain period of time after administration of the antibody or fragment thereof, that is to say that administration of the antibody to the subject may completely prevent the subject from developing a migraine for e.g. at least 1 , 2, 3, 4, 5 or 6 days, 1 , 2, 3, 4, 5 or 6 weeks or 1 , 2, 3, 4, 5 or 6 months. If the therapy completely prevents the subject from developing a migraine for a certain period of time after administration of the antibody, in that time the subject does not develop a migraine, and may experience no migraine symptoms (e.g. pain or aura).
  • Preventing migraine also includes reducing the frequency of migraines suffered by the patient.
  • the therapy provided herein may reduce the frequency of migraine or migraine days per months suffered by the patient by e.g. at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 % for a certain period of time after administration of the antibody or fragment thereof (relative to the frequency of migraines suffered by the patient prior to receiving the therapy).
  • the reduction in migraine frequency in the subject may last for e.g. at least 1 , 2, 3, 4, 5 or 6 days, 1 , 2, 3, 4, 5 or 6 weeks or 1 , 2, 3, 4, 5 or 6 months.
  • a patient suffering migraines with a reduced frequency after receiving the therapy described herein may suffer migraines about, or no more than, once every 1 , 2, 3, 4, 5 or 6 weeks or 1 , 2, 3, 4, 5 or 6 months.
  • treating migraine is meant that the methods provided herein are applied to the subject (i.e. the antibody or fragment thereof is administered to the subject) when the subject is experiencing migraine symptoms and causes an improvement in (i.e. reduction in or termination of) the symptoms.
  • the antibody or fragment thereof may be administered to the subject at any point during a migraine, particularly at or around the onset of symptoms, though it may be later in the course of the migraine, and causes the migraine symptoms to become less severe or to disappear entirely.
  • migraine treatment encompasses both complete and partial relief of symptoms of an ongoing migraine.
  • the therapy may achieve a reduction in the severity of symptoms of at least e.g. 10, 20, 30, 40, 50, 60, 70, 80 or 90 %, as assessed by the subject or their physician. Any migraine symptom may be relieved by the therapy provided herein, e.g. headache.
  • the antibody or fragment thereof may be administered to a subject at the first hint of migraine symptoms in the subject, and prevent the onset of a migraine. Such a case may be seen as both treating and preventing a migraine.
  • Alleviating migraine is meant that the therapy provided herein causes a partial, but not complete, reduction in the severity of migraine symptoms in a subject. Alleviation of a migraine may be achieved by administering the antibody or fragment thereof to the subject when the subject is suffering from a migraine (as discussed above in the treatment section). In this case, alleviation of the migraine would be achieved if a partial reduction in the severity of the migraine symptoms results from the therapy. Such a partial reduction may be as described above, i.e. a reduction in the severity of symptoms of at least e.g. 10, 20, 30, 40, 50, 60, 70, 80 or 90 %, as assessed by the subject or their physician.
  • migraine-related disability is disability caused by migraines.
  • a subject may be considered to suffer from migraine-related disability if their migraines prevent them from undertaking daily activities which would otherwise be normal for a person of the age of the subject.
  • migraine-related disability may result in an inability to work, or at least cause the subject to have to reduce their working hours.
  • Migraine-related disability may result in an inability to study or receive education or training.
  • Migraine-related disability may result in an inability to perform housework or care for oneself or ones children, or in an inability to socialise or undertake hobbies.
  • migraine migraine-related disability
  • end the subject migraine-related disability
  • the migraine condition of the subject is no longer disabling.
  • the subject may gain the ability to return to work, or increase their working hours.
  • the subject may gain the ability to restart hobbies, to exercise or play sport, to perform housework, to care for children or other dependents, or generally improve their ability to perform normal daily activities.
  • the subject’s quality of life may as a result improve, as may be measured qualitatively or quantitively by score-based assessment or suchlike.
  • a reduction in the duration of the migraine symptoms may also be seen as an alleviation of the migraine.
  • the duration of migraine symptoms may be reduced by the therapy provided herein by e.g. at least about 10, 20, 30, 40, 50, 60, 70, 80 or 90 %, e.g. at least 2, 4, 6, 8, 10, 12, 18, 24, 36 or 48 hours or more.
  • the antibody or fragment thereof is administered as a preventative therapy, and does not completely prevent the subject from developing migraines, but does reduce the frequency with which the subject develops migraines and/or the severity or length of the migraines which do occur, this can be considered alleviation of migraines.
  • a reduction in frequency, severity or length of migraines achieved by the therapy provided herein may be of any degree, as discussed above. As is apparent, there is overlap between “treatment”, “alleviation” and “prevention” of migraines as defined herein, and thus the therapy provided herein may treat, alleviate and/or prevent migraines in a subject.
  • the subject to whom the therapy provided herein is administered may be any subject in need of such therapy.
  • the subject is a subject who suffers from migraines, particularly a subject who suffers from frequent and/or debilitating migraines (e.g. migraines of a particularly long duration).
  • the subject may suffer migraines at least about once a month, every 3 weeks, every 2 weeks or every week, or about 2, 3, 4 times a week or more.
  • the subject may be impacted by migraine symptoms for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days per month (that is to say, 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 on at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days per month.
  • the antibody or fragment thereof may be administered to the subject as a preventive therapy, or as active therapy for an ongoing migraine.
  • the subject is a human, but may be a non-human animal adjudged by a veterinarian to be suffering from migraines.
  • the subject may be a domestic animal, particularly a companion animal, e.g. a dog, cat, horse or suchlike.
  • the subject suffers from migraines which are not responsive to therapy with an inhibitor of calcitonin gene-related peptide (CGRP).
  • CGRP calcitonin gene-related peptide
  • the subject is intolerant to an inhibitor of CGRP.
  • CGRP inhibitor An inhibitor of CGRP may be referred to as a CGRP inhibitor, and the terms “CGRP inhibitor” and “inhibitor of CGRP” are used interchangeably herein.
  • a CGRP inhibitor as defined herein is an agent which blocks the activity of CGRP, and in particular which prevents CGRP from activating the CGRP receptor. CGRP inhibitors are described further below.
  • aCGRP therapy includes antagonists to the CGRP receptor and ligand, including both mAbs and non-peptide small molecules (gepants) used for acute and preventative treatment.
  • anti-CGRP (aCGRP) therapy and “CGRP inhibitor” and “inhibitor of CGRP” may be used interchangeably herein.
  • the subject may suffer from migraines which are not responsive to therapy with a CGRP inhibitor.
  • a subject generally suffers from frequent and/or recurrent migraines, and has received therapy with a CGRP inhibitor.
  • the subject has received at least one prior treatment for migraine, namely a CGRP inhibitor.
  • the subject may have received multiple prior 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 instance, the subject may have tried multiple different dosage levels of one or more CGRP inhibitors, multiple different dosage frequencies, different administration routes of one or more CGRP inhibitors, etc.
  • a subject is not responsive (or non-responsive) to therapy with a CGRP inhibitor this means that the subject has found that a CGRP inhibitor does not effectively treat, alleviate or prevent their migraines.
  • a subject who suffers from migraines which are not responsive to therapy with a CGRP inhibitor may find that their condition has not improved during or following the CGRP inhibitor therapy, i.e. that their migraines have not become less severe, shorter or less frequent in response to therapy with a CGRP inhibitor. That is to say, the subject may be a subject for (or in) whom CGRP inhibitor therapy was completely ineffective for migraine treatment. In this case the subject’s condition may not have improved at all, i.e. the severity, duration and frequency of the subject’s migraines during and after the CGRP inhibitor therapy may be unchanged relative to before they received the CGRP inhibitor therapy.
  • the CGRP inhibitor e.g. painkillers such as paracetamol or ibuprofen or other migraine treatments as discussed below
  • the CGRP inhibitor has provided no additional benefit above any benefit achieved by the other drug(s).
  • a subject’s condition may even have deteriorated in response to CGRP inhibitor therapy, e.g. the severity, duration and/or frequency of the subject’s migraines may have worsened.
  • the subject may have seen only a minor or partial improvement in their condition in response to CGRP inhibitor therapy, e.g. a minor or partial reduction in migraine severity, duration and/or frequency.
  • a minor or partial reduction in migraine severity, duration and/or frequency e.g. a minor or partial reduction in migraine severity, duration and/or frequency.
  • the subject in response to CGRP inhibitor therapy the subject may have seen an improvement (i.e. reduction) in monthly migraine days (in case of episodic migraine) or monthly headache days (in case of chronic migraine) frequency of less than 30 % (e.g. less than 25 %, 20 %, 15 %, 10 % or 5 %) and/or remain adversely affected on 2, 3, 4, or 5 or more days per month due to their migraine severity, duration and/or frequency.
  • the subject is taking or being administered one or more additional therapies for their migraines at the same time as the CGRP inhibitor, in this case the minor improvement is seen above any benefit provided by the additional therapies.
  • a subject may be considered by a physician to have responded poorly to CGRP inhibitor therapy, e.g. the subject may have responded less well to CGRP therapy than the physician may have expected or hoped.
  • the subject may be considered by a physician to require an alternative line of therapy to CGRP inhibitor therapy in order to treat, alleviate or prevent their migraines.
  • the subject may have found that their condition had improved slightly as a result of CGRP inhibitor therapy, but not to a degree which had improved their quality of life or enabled them to undertake normal daily activities.
  • the subject may have found (depending on their initial condition) that despite receiving CGRP inhibitor therapy they were still not able to work, to increase their working hours, to perform housework, or to go out or socialise, etc.
  • the subject’s migraines may have become non-responsive to therapy with a CGRP inhibitor.
  • therapy with a CGRP inhibitor may have initially been successful in treating, alleviating or preventing the subject’s migraines (as set out above), but after the initial response the subject’s migraines may have returned, or worsened, e.g. returned to their original severity, duration and/or frequency. That is to say, the subject’s migraines may have become refractory to therapy with a CGRP inhibitor, i.e. the CGRP inhibitor may have lost efficacy in the subject.
  • Lack of efficacy, partial efficacy or loss of efficacy of a CGRP inhibitor may be selfdiagnosed by a subject or diagnosed by the subject’s physician. In the case of non-human subjects, lack of efficacy or partial efficacy may be diagnosed by the subject’s owner, keeper, vet or such person as is able to monitor the condition of the subject.
  • the subject may be a CGRP-lnadequate Responder (aCGRP-IR), also referred to as an Inadequate Responder to anti-CGRP therapy.
  • aCGRP-IR CGRP-lnadequate Responder
  • the subject has failed to respond to > 3 adequately dosed (maximum tolerated dose for > 2 months) small molecule migraine preventive treatments from different classes and who have failed one or more aCGRP therapies (aCGRP-inadequate responder [aCGRP-IR]).
  • failure is defined as having no clinically meaningful improvement per the treating physician’s judgement or having discontinued aCGRP therapy due to AEs that made treatment intolerable.
  • migraines may be CGRP-dependent or CGRP-independent. Without being bound by theory, it is believed that CGRP-dependent migraines are caused by signalling through the CGRP/CGRP receptor axis as described above, whereas CGRP-independent migraines have an unknown mechanism which does not rely on signalling through the CGRP/CGRP receptor axis. It has previously been shown that PAR2 inhibition is effective in preventing migraine in a mouse model of CGRP-dependent migraine (Kopruszinski et al., Cephalagia 40(14) 15-35-1550, 2020), but the present inventors are the first to demonstrate that PAR2 inhibition is effective in preventing CGRP-independent migraine.
  • Migraines which are refractory (or resistant) to treatment with CGRP inhibitors may be CGRP-independent migraines.
  • CGRP inhibitor therapy cannot be expected to be effective, as signalling through the CGRP/CGRP receptor axis does not play a causative role in the migraines.
  • PAR2 inhibition therapy is a suitable and effective alternative for such subjects.
  • the therapy provided herein can be seen as a method for treating, alleviating or preventing CGRP-independent migraines.
  • there is no means of determining whether a migraine patient is suffering from CGRP-dependent or CGRP-independent migraines other than administering a CGRP inhibitor to the patient and assessing its efficacy.
  • Patients in whom a CGRP inhibitor is an effective migraine therapy can thus be seen to be suffering from CGRP-dependent migraines, and patients in whom a CGRP inhibitor is an ineffective migraine therapy can be seen to be suffering from CGRP-independent migraines.
  • a patient may be suffering concurrently from both CGRP-dependent and CGRP-independent migraines.
  • a CGRP inhibitor would be expected to be partially effective as a migraine therapy, as the CGRP inhibitor would in this instance block the CGRP-dependent contribution to the patient’s migraines, but not the CGRP- independent contribution.
  • such a patient may gain a minor or partial improvement in the severity, duration and/or frequency of their migraines from therapy with a CGRP inhibitor, but will experience much less benefit than a patient whose migraines are purely CGRP-dependent.
  • Such a patient will benefit from therapy in accordance with the methods provided herein in order to block the CGRP-independent contribution to their migraines.
  • the subject may be intolerant to a CGRP inhibitor and therefore unable to receive CGRP inhibitor therapy.
  • therapy with a PAR2 inhibitor provides a suitable alternative to CGRP inhibitor therapy.
  • a subject is deemed intolerant to a CGRP inhibitor if the subject cannot receive CGRP inhibitor therapy for a reason unrelated to efficacy. That is to say, a subject who is intolerant to a CGRP inhibitor may be any subject for whom migraine therapy with a CGRP inhibitor is contraindicated for a reason other than lack of efficacy. For instance, 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 which causes it to be unsafe for the subject to take or be administered a CGRP inhibitor, or may be taking one or more other drugs which are incompatible with a CGRP inhibitor. Intolerance to a particular CGRP inhibitor may be routinely diagnosed by a physician.
  • a subject may be intolerant to one CGRP inhibitor or multiple CGRP inhibitors (i.e. more than one CGRP inhibitor).
  • a subject may be intolerant to one or more classes of CGRP inhibitor.
  • the subject has tried multiple different CGRP inhibitors, the subject has found themselves non-responsive to treatment with and/or intolerant to every CGRP inhibitor tried.
  • the subject may be any animal suffering from migraines.
  • the animal thus may be a human, or may not.
  • the subject is either non-responsive to or intolerant of therapy with an inhibitor of CGRP of the species of the subject.
  • a human subject to be treated as described herein is either non- responsive to migraine therapy with an inhibitor of human CGRP or is intolerant (e.g. allergic) to an inhibitor of human CGRP.
  • first and second line migraine treatments exist which at present are commonly prescribed before a CGRP inhibitor, which is usually seen as a third line treatment.
  • the 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 treatment with or intolerant of a CGRP inhibitor).
  • the subject may be non-responsive to therapy with a first line migraine treatment.
  • the subject may be non-responsive to therapy with a second line migraine treatment.
  • the subject may be non-responsive to therapy with a first line migraine treatment and non- responsive to therapy with a second line migraine treatment.
  • First line migraine treatments include triptans, p-blockers, topiramate (a carbonic anhydrase inhibitor) and candesartan (an angiotensin receptor blocker).
  • triptans which may 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, such as one or more of the triptans listed above.
  • Examples of p-blockers which may 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 p-blockers, such as one or more of the P-blockers listed above.
  • the subject may be non-responsive to migraine therapy with candesartan.
  • the subject may be non-responsive to therapy with topiramate.
  • the subject may be non-responsive to migraine therapy with one first-line treatment (as set out above) or multiple first-line treatments.
  • the subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with one or more p-blockers.
  • the subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with candesartan.
  • the subject may be non-responsive to migraine therapy with one or more p-blockers and non-responsive to migraine therapy with candesartan.
  • the subject may be non-responsive to migraine therapy with one or more triptans and non-responsive to migraine therapy with topiramate.
  • the subject may be non-responsive to migraine therapy with one or more P-blockers and non-responsive to migraine therapy with topiramate.
  • the 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 one or more triptans, non-responsive to migraine therapy with one or more p-blockers and non- responsive to migraine therapy with candesartan.
  • the subject may be non-responsive to migraine therapy one or more triptans, non-responsive to migraine therapy with one or more P-blockers and non-responsive to migraine therapy with topiramate.
  • the subject may be non-responsive to migraine therapy 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 p-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 P-blockers, non-responsive to migraine therapy one or more triptans, non-responsive to migraine therapy with topiramate and non-responsive to migraine therapy with candesartan. That is to say, the subject may be non-responsive to migraine therapy with one or more triptans, one or more p-blockers, topiramate and/or candesartan.
  • Second line migraine treatments include calcium channel blockers, tricyclic antidepressants, and valproic acid and salts thereof.
  • calcium channel blockers which may 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 which may be used as second line migraine treatments include am itryptiline, 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.
  • tricyclic antidepressants such as one or more of the tricyclic antidepressants listed above.
  • salts of valproic acid which may be used as second line migraine treatments include sodium valproate.
  • the subject may be non-responsive to migraine therapy with valproic acid or a salt thereof, such as sodium valproate.
  • the subject may be non-responsive to migraine therapy with one second-line treatment (as set out above) or multiple second-line treatments.
  • 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 to say, 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 (e.g. sodium valproate).
  • valproic acid or a salt thereof e.g. sodium valproate
  • the subject may be non-responsive to migraine therapy with a first and/or second line migraine treatment.
  • the subject may be non-responsive to one or more of the first line migraine treatments as set out above, and/or non-responsive to one or more of the second line migraine treatments as set out above.
  • the subject may be non-responsive to migraine therapy with one or more triptans, one or more p-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).
  • the subject to be treated according to the methods provided herein thus may have previously been treated with at least one first line migraine therapy as set out above, and/or at least one second line migraine therapy as set out above, in which case the treatment with the first and/or second migraine therapy was unsuccessful. That is to say, therapy with a first and/or second line migraine treatment agent did not effectively treat, alleviate or prevent the subject’s migraines.
  • ‘Non-responsiveness’ to therapy with a first and/or second line migraine treatment agent thus has an equivalent or corresponding meaning to non-responsiveness to therapy with a CGRP inhibitor, as described above, i.e. that treatment of the subject with a first and/or second line migraine therapy lacked efficacy, was only partially efficacious or lost efficacy.
  • the subject may be intolerant to a first and/or second line migraine therapy, as described above.
  • ‘Intolerance’ to a first or second line migraine therapy has an equivalent or corresponding meaning to intolerance to a CGRP inhibitor, as described above, i.e. that the subject cannot receive treatment with that first or second line migraine therapy for a reason unrelated to efficacy, e.g. for the reasons set out above in relation to intolerance to a CGRP inhibitor.
  • the subject has tried more than one first or second line migraine therapy, the subject has found themselves non-responsive to treatment with or intolerant to each first or second line migraine therapy tried.
  • the subject may have overused or be overusing acute medication in an attempt to control their migraine condition.
  • Acute medication includes over-the-counter painkillers, such as paracetamol and ibuprofen, and also first and second line treatments which are taken in response to migraine symptoms rather than administered according to a defined dosing schedule (e.g. triptans).
  • overuse of such acute medication may mean that the subject has taken or is taking more than the maximum recommended dose of the medication, or that the subject is using the medication excessively frequently. If the subject is taking acute medication in such doses that it is causing side effects or damaging (or risking damaging) the general health or wellbeing of the subject, such use is considered overuse.
  • the subject to be treated is human and the CGRP inhibitor is thus a human CGRP inhibitor. That is to say, the subject may be non-responsive to migraine therapy with, or intolerant to, an inhibitor of human CGRP.
  • a CGRP inhibitor as defined herein is an agent which blocks the activity of CGRP, and in particular which prevents CGRP from activating the CGRP receptor.
  • Human CGRP has two different forms: aCGRP (SEQ ID NO: 12) and pCGRP (SEQ ID NO: 13).
  • aCGRP is encoded by the CALCA gene and pCGRP by the CALCB gene. The same may also be true in other animals.
  • the two forms of human CGRP share over 90 % sequence identity, differing by only 3 amino acids, and have essentially equivalent biological activity, but aCGRP is the principal form found in the central and peripheral nervous system, whereas pCGRP is found mainly in the enteric nervous system (Russell et al., Physiological Reviews 94(4): 1099-1142, 2014). Together, the aCGRP and pCGRP peptides are referred to as CGRP.
  • a CGRP inhibitor as defined herein may be an inhibitor of aCGRP (particularly human aCGRP) or may be an inhibitor of pCGRP (particularly human pCGRP), but preferably, and generally, is an inhibitor of both aCGRP and pCGRP. That is to say, a CGRP inhibitor as defined herein may prevent aCGRP from activating the CGRP receptor, or may prevent pCGRP from activating the CGRP receptor, but preferably, and generally, prevents aCGRP and pCGRP from activating the CGRP receptor.
  • the CGRP receptor comprises two proteins: CLR and RAMP1 .
  • the human CLR protein has the UniProt accession number Q16602
  • the human RAMP1 protein has the UniProt accession number 060894.
  • the CGRP inhibitor to which the subject is either intolerant and/or non-responsive may be an antibody.
  • the antibody prevents CGRP from binding to and/or activating the CGRP receptor.
  • such an antibody binds either CGRP or the CGRP receptor. If the antibody binds the CGRP receptor it may bind either the CLR protein or the RAMP1 protein.
  • An antibody which is a CGRP inhibitor may prevent CGRP from activating the CGRP receptor by blocking binding of CGRP to the CGRP receptor.
  • Such an antibody may act sterically by binding CGRP at or near the location to which it binds the CGRP receptor, or by binding the CGRP receptor at or near the location to which it binds CGRP, such that the CGRP-CGRP receptor interaction is physically blocked.
  • binding of CGRP to the CGRP receptor may be inhibited by an antibody which binds CGRP or the CGRP receptor at a site which is distant to the CGRP-CGRP receptor interface, but which causes a change in shape or conformation of CGRP or the CGRP receptor such that binding of CGRP to the CGRP receptor is inhibited.
  • an antibody which is a CGRP inhibitor may not prevent CGRP from binding the CGRP receptor but instead inhibit activation of the CGRP receptor in response to CGRP binding.
  • an antibody may bind the CGRP receptor and lock it in an inactive conformation, such that binding of CGRP cannot activate the receptor.
  • CGRP inhibitors include: erenumab (Amgen/Novartis), which binds the CGRP receptor; fremanezumab (Teva), which binds CGRP; galcanezumab (Eli Lilly), which binds CGRP; and eptinezumab (Lundbeck), which binds CGRP.
  • the subject treated according to the methods herein may be non-responsive to migraine treatment with, or intolerant to, an antibody that inhibits CGRP, in particular an antibody that binds CGRP or the CGRP receptor and prevents binding of CGRP to the CGRP receptor, or prevents activation of the CGRP receptor.
  • the subject may be non-responsive to migraine treatment with, or intolerant to, erenumab, fremanezumab, galcanezumab and/or eptinezumab.
  • the CGRP inhibitor to which the subject is either intolerant and/or non- responsive may be a small molecule.
  • a small molecule inhibitor of CGRP may act to prevent activation of the CGRP receptor by CGRP in any way, as set out above in respect of a CGRP inhibitor antibody.
  • a small molecule inhibitor of CGRP binds either CGRP or the CGRP receptor, and acts directly or indirectly to block binding of CGRP to the CGRP receptor, or does not inhibit binding of CGRP to the CGRP receptor but inhibits activation of the CGRP receptor in response to binding of CGRP thereto.
  • CGRP inhibitors include gepants, which bind the CGRP receptor and inhibit its activation.
  • gepants include rimegepant, ubrogepant, zavegepant, olcegepant and atogepant.
  • the subject treated according to the methods herein may be non-responsive to migraine treatment with, or intolerant to, one or more gepants.
  • the subject may be non-responsive to migraine treatment with, or intolerant to, rimegepant, ubrogepant, zavegepant, olcegepant and/or atogepant.
  • the subject treated according to the methods herein may be non-responsive to migraine treatment with, or intolerant to, gepants, that is to say the subject treated according to the methods herein may be non-responsive to migraine treatment with, or intolerant to, the gepant class of small molecules.
  • the subject may be non-responsive to migraine treatment with, or intolerant to, any other therapy or therapy type targeting the CGRP-CGRP receptor signalling axis.
  • the subject may be non-responsive to migraine treatment with, or intolerant to, a large molecule therapeutic which inhibits CGRP, e.g. a peptide or nucleic acid drug which inhibits CGRP, e.g. by the mechanisms set out above in respect of antibodies and small molecules, or gene therapy which acts to inhibit CGRP activity (e.g. by knocking out or knocking down CGRP or the CGRP receptor).
  • the subject may be non-responsive to migraine therapy with, and/or intolerant to, one or more classes of CGRP inhibitor.
  • the subject may be non-responsive to migraine therapy with, and/or intolerant to, both antibodies and small molecules that inhibit CGRP, in particular the subject may be non-responsive to migraine therapy with, and/or intolerant to, antibodies which inhibit CGRP and gepants.
  • an antibody is a protein comprising two heavy chains and two light chains.
  • the light chains are shorter (and thus lighter) than the heavy chains.
  • the heavy chains comprise an N-terminal heavy chain variable domain (V H )
  • the light chains comprise an N-terminal light chain variable domain ( L). Both chains comprise constant domains C-terminal to the variable domain.
  • the specificity of an antibody is determined by the sequence of its variable region.
  • Both the light and heavy chains of an antibody comprise three hypervariable complementarity-determining regions (CDRs), such as those set out herebelow.
  • CDRs complementarity-determining regions
  • the CDR sequences determine the specificity of an antibody.
  • the three CDRs of a heavy chain are known as VHCDR1 , VHCDR2 and VHCDR3, from N-terminus to C-terminus
  • the three CDRs of a light chain are known as VLCDR1 , VLCDR2 and VLCDR3, from N-terminus to C-terminus.
  • Antigen-binding fragments of antibodies are fragments or synthetic constructs comprising one or more antigen-binding sites of an antibody, but not the entire antibody.
  • an antigen-binding fragment of an antibody comprises the entire V L and V H domain sequences, but lacks the heavy and light chain constant domains, and does not contain the entirety thereof.
  • the antibody (or fragment thereof) for use in the methods provided herein specifically binds to PAR2.
  • the antibody, or fragment thereof may bind PAR2 from any species of interest.
  • the antibody or fragment thereof binds PAR2 from the species to which the subject belongs.
  • the antibody or fragment thereof may bind e.g. canine, feline or equine PAR2.
  • the antibody or fragment thereof specifically binds human PAR2.
  • the amino acid sequence of human PAR2 has the UniProt accession no. P55085, and is also set out in SEQ ID NO: 14. If the antibody used herein specifically binds to human PAR2 it may bind human PAR2 but not PAR2 from other, nonhuman species, or may bind to human PAR2 and PAR2 from other, non-human species.
  • an antibody which binds specifically to human PAR2 is an antibody which binds to human PAR2 with a greater affinity than that with which it binds to other molecules (e.g. with an affinity as described below), or at least most other molecules.
  • the antibody binds to a sequence or configuration present on human PAR2, preferably a unique sequence or configuration not present on other molecules.
  • an antibody which specifically binds human PAR2 does not necessarily bind only to human PAR2: the antibody may cross-react with certain other undefined target molecules, or may display a level of non-specific binding when contacted with a mixture of a large number of molecules (such as a cell lysate or suchlike).
  • an antibody which specifically binds human PAR2 may display cross-reactivity with PAR2 from other species, e.g. those mentioned above.
  • the skilled person can readily determine whether an antibody or fragment thereof specifically binds human PAR2 using standard techniques in the art, e.g. ELISA, Western-blot, surface plasmon resonance (SPR), etc.
  • the antibody or fragment thereof for use herein may bind PAR2 in a pH-dependent manner.
  • the anti-PAR2 antibody or antigen-binding fragment thereof for use herein may bind PAR2 with high affinity, e.g. with a K D of less than about 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 200 pM, 100 pM or 50 pM.
  • the high affinity binding preferably occurs at physiological, extracellular pH (i.e. about pH 7.4).
  • the antibody or antigen-binding fragment thereof for use herein may bind PAR2 with a slightly lower affinity at a slightly acidic pH (such as pH 6.0) relative to at physiological, extracellular pH. That is to say, the antibody or fragment thereof may bind to PAR2 with a higher (or greater) affinity at pH 7.4 than it does at pH 6.0.
  • the antibody or fragment thereof for use herein may bind PAR2 with a K D of greater than about 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.
  • the K D with which the antibody or fragment thereof binds PAR2 at any given pH may be determined using standard methods in the art, e.g. SPR.
  • the anti-PAR2 antibody or antigen-binding fragment thereof for use herein may specifically bind to PAR2 with a dissociative half-life (ti/2) of greater than about 1.5 minutes, 1.75 minutes, 2 minutes, 2.5 minutes, 3 minutes, 5 minutes, 10 minutes, 20 minutes, or 30 minutes as measured using an assay such as surface plasmon resonance at pH 7.4, at a temperature of e.g. 25°C or 37°C.
  • the anti-PAR2 antibody or antigen-binding fragment thereof may bind to PAR2 with a dissociative half-life (ti/ 2 ) of less than about 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 13 seconds, 7 seconds, 5 seconds, or 3 seconds as measured using an assay such as surface plasmon resonance at a slightly acidic pH (e.g., pH 6), at a temperature of e.g. 25°C or 37°C.
  • a dissociative half-life ti/ 2
  • ti/ 2 dissociative half-life of less than about 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 13 seconds, 7 seconds, 5 seconds, or 3 seconds as measured using an assay such as surface plasmon resonance at a slightly acidic pH (e.g., pH 6), at a temperature of e.g. 25°C or 37°C.
  • the antibody or fragment thereof for use in the methods provided herein inhibits the activity of PAR2. That is to say, the antibody or fragment thereof used herein may be referred to as a neutralising anti-PAR2 antibody or an antagonistic anti-PAR2 antibody.
  • a neutralising anti-PAR2 antibody may neutralise PAR2 activity by, e.g. (i) interfering with the interaction between PAR2 and a protease (e.g., trypsin, tryptase and/or matriptase); (ii) inhibiting the cleavage of PAR2 by a protease; or (iii) inhibiting PAR2 signalling or PAR2 activation.
  • the antibodies or antigen-binding fragments thereof may inhibit conversion of inactive, uncleaved PAR2 into active, cleaved PAR2, and/or inhibit exposure of the tethered ligand.
  • the antibodies or antigen-binding fragments thereof may inhibit activation of PAR2 by its tethered ligand, e.g. by inhibiting binding of the tethered ligand to the second transmembrane domain of PAR2 (which as noted above is required for PAR2 activation).
  • the antibody or fragment thereof for use in the methods provided herein may inhibit PAR2 activity by inhibiting the cleavage of the PAR2 extracellular domain.
  • cleavage of the PAR2 extracellular domain by serine proteases results in PAR2 activation, and thus prevention of the cleavage of this domain serves to inhibit PAR2 activation.
  • the antibody (or fragment thereof) may thus prevent proteolytic cleavage of the PAR2 extracellular domain by a serine protease.
  • the antibody (or fragment thereof) may prevent cleavage of the PAR2 extracellular domain by trypsin, tryptase or matriptase.
  • Other proteases which may act to cleave the PAR2 extracellular domain include elastase and proteinase 3.
  • the antibody may inhibit PAR2 cleavage by either of these enzymes, and any other protease enzyme capable of cleaving PAR2.
  • An antibody may inhibit PAR2 cleavage by e.g. binding PAR2 at or close to its cleavage site, thereby blocking protease access to the cleavage site.
  • the antibody or antigen-binding fragment thereof may prevent proteases from interacting with PAR2, and in particular may prevent trypsin, tryptase and/or matriptase from interacting with PAR2, thereby inhibiting cleavage and the resulting activation of PAR2.
  • the antibody or fragment thereof for use herein thus may bind the intact (uncleaved) extracellular domain of PAR2, i.e.
  • the antibody or fragment thereof may bind PAR2 at an epitope located on its extracellular domain prior to cleavage (such an epitope may also be present in the extracellular domain after its cleavage.
  • the important feature of such an epitope located on the PAR2 extracellular domain prior to cleavage is that binding of an antibody to that epitope inhibits proteolytic cleavage of the PAR2 extracellular domain, e.g. by blocking protease access to the PAR2 cleavage site).
  • the anti-PAR2 antibody or fragment thereof may block the interaction between PAR2 and a protease (e.g. trypsin) in vitro with an IC 5 o value of less than about 15 nM, as measured by a binding assay such as those described in WO 2018/167322 (incorporated herein by reference).
  • a protease e.g. trypsin
  • the antibody or antigen-binding fragment may block the interaction between PAR2 and a protease (e.g.
  • trypsin in vitro at a pH of about 7.4 with an IC 5 o value of less than about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 500 pM, 400 pM, 200 pM, 100 pM, 50 pM, 5 pM, 1 pM or 0.1 pM.
  • the antibody or fragment thereof may block the interaction between PAR2 and a protease (e.g. trypsin) in vitro at a pH of about 6.0 with an IC 5 o value of greater than about 300nM, 500 nM, 750 nM, 1000 nM, 1100 nM, or 1200 nM.
  • cleavage of the PAR2 extracellular domain causes exposure of the PAR2 tethered ligand, a PAR2 sequence which binds the PAR2 activation site, thereby activating the receptor.
  • the antibody or fragment thereof for use herein may thus inhibit PAR2 activity by inhibiting exposure of the PAR2 tethered ligand, or 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 thus the antibody or fragment thereof may prevent the PAR2 tethered ligand from interacting with the extracellular loop 2 domain of PAR2.
  • Inhibition of exposure of the PAR2 tethered ligand, or its interaction with the extracellular loop 2 domain of PAR2, may of course be achieved by preventing cleavage of PAR2 in the first place, as set out above.
  • the antibody may block exposure of the tethered ligand, or its interaction with the PAR2 activation site, after cleavage of the PAR2 extracellular domain.
  • the antibody or fragment thereof may bind the PAR2 tethered ligand, thereby blocking its exposure and preventing it from interacting with the PAR2 activation site.
  • the human PAR2 tethered ligand has the amino acid sequence set out in SEQ ID NO: 15.
  • the antibody or fragment thereof used herein may bind PAR2 at an epitope within SEQ ID NO: 15, or an amino acid sequence having at least 80, 85, 90 or 95 % sequence identity thereto.
  • the antibody or fragment thereof for use herein may bind the PAR2 activation site, thereby blocking the interaction between the activation site and the tethered ligand.
  • 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), and accordingly the antibody or fragment thereof may bind 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).
  • the antibody or fragment thereof for use herein may bind PAR2 at an epitope located within SEQ ID NO: 16, or amino acid sequence having at least 80, 85, 90 or 95 % sequence identity thereto.
  • the antibody or fragment thereof for use herein inhibits the activity of PAR2. This inhibition may be seen as inhibition of the activation of PAR2. As detailed above, this inhibition may be achieved by any mechanism which has the effect of inhibiting PAR2 activity.
  • the inhibition caused by the anti-PAR2 antibody or fragment thereof for use herein may be complete (i.e. PAR2 activity is completely inhibited, such that the receptor is completely inactivated by binding of the antibody thereto). However, the inhibition need not be complete so long as it is detectable using an appropriate assay, and is sufficient to achieve successful treatment, alleviation or prevention of migraine in subjects to whom it is administered.
  • the antibody or antigen-binding fragment thereof may inhibit PAR2 activity by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 % or 90 %, or by 100 %, as compared to uninhibited active PAR2. Higher degrees of inhibition are advantageous as such antibodies may achieve superior therapeutic results than antibodies with a lesser inhibitor effect, or may achieve the same results at lower doses.
  • assays for detecting activity of an anti-PAR2 antibody or antigenbinding fragment thereof in PAR2 inhibition are described in the Examples of WO 2018/167322.
  • PAR2 inhibition may be measured using a cell-based assay, in particular a calcium flux assay.
  • PAR2 activation is known to increase the intracellular Ca 2+ concentration (as a result, it is believed, of mobilising intracellular Ca 2+ stores, rather than causing Ca 2+ influx into the cell from outside of the cell) and so a change in intracellular Ca 2+ concentration can be measured as a proxy for PAR2 activation.
  • Cells which endogenously express PAR2 e.g. human A549 cells, cynomolgus CYNOM-K1 cells, rat KNRK cells or murine LL/2 cells
  • PAR2-expressing cells are first loaded with a calcium dye (e.g. Screen QuestTM 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 desensitise PAR1 activity. Trypsin may then be applied to the cells and calcium responses measured based on the activity of the calcium dye.
  • a calcium dye e.g. Screen QuestTM Fluo-8 dye, AAT Bioquest (USA)
  • thrombin e.g. Screen QuestTM Fluo-8 dye, AAT Bioquest (USA)
  • Trypsin may then be applied to the cells and calcium responses measured based on the activity of the calcium dye.
  • the Fluo-8 dye mentioned above and exemplified in WO 2018/167322 is a fluorescent dye, the fluorescence of which is enhanced by calcium binding. The change in fluorescence in response to application of trypsin to the cells indicates the degree of PAR2 activity caused by the trypsin.
  • the anti-PAR2 antibody or fragment thereof used herein may cause a reduction of at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 %, or even a reduction of 100 %, in the trypsin response by PAR2-expressing cells in a calcium flux assay, as described above.
  • the antibody or antigen-binding fragment thereof used herein may inhibit calcium influx in a calcium flux assay (e.g. as described above) with an IC 5 o of less than 1 nM, 500 pM, 400 pM, 200 pM, 100 pM, 50 pM, 10 pM, 5 pM, 1 pM or 0.1 pM.
  • the antibody or antigen-binding fragment thereof used herein may have an IC50 of less than 5.0 x 10 10 M, 4.5 x 10 10 M, 4.0 x 10 10 M, 3.5 x 10 10 , 3.0 x W 10 M, 2.5 x 10 10 M, 2.0 x 10' 10 M, 1.5 x 10' 10 M or 1.0 x 10 -10 M in a calcium flux assay in human A549 cells.
  • the antibody or antigen-binding fragment thereof used herein may have an IC50 of less than 10 9 M, 9.5 x 10 10 M, 9.0 x 10 -10 M, 8.5 x 10 10 M, 8 x 10 10 M, 7.5 x 10 10 M, 7.0 x W 10 M,
  • the antibody or antigen-binding fragment thereof used herein may have an IC50 of less than IO 10 M, 9.5 x 10TM M, 9.0 x 10TM M, 8.5 x 10TM M, 8 x 10TM M, 7.5 x 10TM M,
  • the antibody or antigen-binding fragment thereof used herein may have an IC50 of less than 10 -10 M, 9.5 x 10TM M, 9.0 x 10TM M,
  • an antibody or an antigen-binding fragment thereof (also referred to herein as an “antibody fragment”) is used in the methods provided herein.
  • An “antibody” is an immunoglobulin having the features described hereinbefore.
  • the antibody or antibody fragment used herein is generally monoclonal, i.e. a monoclonal antibody or monoclonal antibody fragment may be used.
  • monoclonal antibody is meant an antibody preparation consisting of a single antibody species, i.e. all antibodies in the preparation have the same amino acid sequences, including the same CDRs, and thus bind the same epitope on their target antigen (by “target antigen” is meant the antigen containing the epitope bound by a particular antibody, i.e. the target antigen of an anti-PAR2 antibody is PAR2 ) with the same effect.
  • a monoclonal antibody fragment means a preparation of an antibody fragment consisting of a single species of antibody fragment. In other words, the antibody or fragment thereof is generally not used herein in the context of or as part of a polyclonal mix of antibodies or antibody fragments.
  • the CDR sequences are located in the variable domains of the heavy and light chains.
  • the CDR sequences sit within a polypeptide framework, which positions the CDRs appropriately for antigen binding.
  • the remainder of the variable domains i.e. the parts of the variable domain sequences which do not form a part of any one of the CDRs
  • the N-terminus of a 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 linking CDR3 to the constant domain forms FR4.
  • variable regions of the antibody used herein may have any suitable sequence such that binding to and inhibition of PAR2 is achieved.
  • the antibody or antigen-binding fragment thereof used herein comprises at least one variable domain (e.g. a H and/or L domain), as necessary to achieve specific binding of PAR2.
  • the antibody or antigen-binding fragment thereof comprises a H and a L domain, though certain antibodies or fragments thereof may require only a single variable domain, particularly a V H domain. For instance, a camelid single domain antibody may be used.
  • antibodies may belong to a number of different isotypes.
  • the isotype of an antibody is determined by the sequence of its constant region.
  • the antibody isotypes are IgG, IgE, IgM, IgA and IgD.
  • Some isotypes may be divided into further subtypes, e.g. there are four sub-types of IgG antibodies: IgG 1 , lgG2, lgG3 and lgG4.
  • the antibody may be of any isotype, i.e. an IgG, IgE, IgM, IgA or IgD antibody may be used.
  • the antibody may be an IgG antibody.
  • an IgG antibody when used it may be of any sub-type, i.e. IgG 1 , lgG2, lgG3 or lgG4 antibody may be used.
  • the antibody may be an IgG 1 antibody.
  • the antibody used herein may comprise a K or A light chain.
  • the antibody or fragment thereof used in the methods herein may be multi-specific, e.g. a bi-specific monoclonal antibody.
  • a multi-specific antibody contains regions or domains (antigen-binding regions) which bind to at least two different molecular binding partners, e.g. bind to two or more different antigens or epitopes.
  • the antibody comprises two heavy and light chains, in the formation as described above, except that the variable domains of the two heavy chains and the two light chains, respectively, are different, and thus form two different antigen-binding regions.
  • a multi-specific e.g.
  • one of the antigen-binding regions has the CDR sequences of an antagonistic anti-PAR2 antibody.
  • the other antigen-binding region(s) of the multispecific antibody is/are different such that they do not act to bind and antagonise PAR2.
  • the other antigen-binding region(s) of the multi-specific antibody for use herein bind a different target, i.e. bind a target other than PAR2.
  • an antigen-binding fragment of an antibody may be used herein.
  • Antibody fragments are discussed in Rodrigo et al., Antibodies, Vol. 4(3), p. 259-277, 2015.
  • Antibody fragments which may 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.
  • a Fab fragment consists of the antigen-binding domain of an antibody, i.e. an individual antibody may be seen to contain two Fab fragments, each consisting of a light chain and its conjoined N-terminal section of the heavy chain.
  • a Fab fragment contains an entire light chain and the V H and C H 1 domains of the heavy chain to which it is bound.
  • Fab fragments may be obtained by digesting an antibody with papain.
  • F(ab')2 fragments consist of the two Fab fragments of an antibody, plus the hinge regions of the heavy domains, including the disulphide bonds linking the two heavy chains together.
  • a F(ab')2 fragment can be seen as two covalently joined Fab fragments.
  • F(ab')2 fragments may be obtained by digesting an antibody with pepsin.
  • F(ab')2 fragments Reduction of F(ab')2 fragments yields two Fab' fragments, which can be seen as Fab fragments containing an additional sulfhydryl group which can be useful for conjugation of the fragment to other molecules.
  • Fv fragments consist of just the variable domains of the light and heavy chains. These are not covalently linked and are held together only weakly by non-covalent interactions. Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. Such a modification is typically performed recombinantly, by engineering the antibody gene to produce a fusion protein in which a single polypeptide comprises both the H and L domains. scFv fragments generally include a peptide linker covalently joining the H and L regions, which contributes to the stability of the molecule.
  • scFv single chain Fv
  • the linker may comprise from 1 to 20 amino acids, such as for example 1 , 2, 3 or 4 amino acids, 5, 10 or 15 amino acids, or other intermediate numbers in the range 1 to 20 as convenient.
  • the peptide linker may be formed from any generally convenient amino acid residues, such as glycine and/or serine.
  • a suitable linker is Gly 4 Ser.
  • Multimers of such linkers may be used, such as for example a dimer, a trimer, a tetramer or a pentamer, e.g. (Gly 4 Ser) 2 , (Gly 4 Ser) 3 , (Gly 4 Ser) 4 or (Gly 4 Ser) 5 .
  • an scFv is herein defined as an antibody fragment, or antigen-binding fragment of an antibody.
  • the antibody fragment used herein may be an analogue of an scFv.
  • the scFv may be linked to other antibodies or fragments thereof (for example other scFvs, Fab antibody fragments and antibodies against different targets).
  • the scFv may be linked to other scFvs so as to form a multimer which is a multi-specific scFv, for example a dimer, a trimer or a tetramer.
  • Bi-specific scFvs are sometimes referred to as diabodies, tri-specific scFvs as triabodies and tetra-specific scFvs as tetrabodies.
  • an scFv may be bound to other, identical scFv molecules, thus forming a multimer which is mono-specific but multi-valent, e.g. a bivalent dimer or a trivalent trimer may be formed.
  • the antibody for use in the methods herein may be a human or humanised antibody (or fragment thereof).
  • Human and humanised antibodies may particularly advantageously be used for the treatment of human subjects.
  • the subject treated according to the methods provided may be a human, and the antibody or fragment thereof used to treat the subject may be human or humanised.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include 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 “humanised” as set out further below.
  • an antigen-binding fragment of a human antibody is an antibody fragment (as set out above) in which the variable domains and, where present, any part of the constant domains present in the fragment, are derived from human germline immunoglobulin sequences.
  • the 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 that are prepared, expressed, created or isolated by recombinant means, such as 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) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • Such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the H and L regions of the recombinant antibodies may be sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody).
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human lgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgGi hinge.
  • Antibodies for use herein may have one or more mutations in the hinge, CH2 or CH3 region (relative to human germline sequences) which may be desirable, for example, in production, to improve the yield of the desired antibody form.
  • the anti-PAR2 antibodies or antigen-binding fragments thereof for use herein may comprise 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 as compared to the corresponding germline sequences from which the antibodies were derived.
  • Human antibodies may be obtained by generating antibodies in a traditional manner by immunising a transgenic mouse with human immunoglobulin genes with the target antigen (i.e. PAR2).
  • Human antibodies may alternatively be obtained by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are described generally in Hoogenboom et al.
  • one method of generating antibodies of interest is through the use of a phage antibody library as described in Lee et al, J. Mol. Biol. (2004), 340(5): 1073-93.
  • synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein.
  • Fv antibody variable region
  • Such phage libraries are panned 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 the nonbinding clones in the library.
  • the 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 provided herein can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences.
  • a “humanised” antibody by contrast is an antibody derived from non-human germline immunoglobulin sequences, but which has been modified to replace non-human sequences with human ones.
  • a humanised antibody may be derived, for instance, from mouse, rat, rabbit, etc., germline immunoglobulin sequences. Indeed, a humanised antibody may be derived from the germline immunoglobulin sequences of any other animal.
  • an antibody is considered humanised if at least one of the H and VL domains is humanised.
  • a humanised antibody may comprise a humanised V H sequence and a humanised V L sequence.
  • a non-human variable domain sequence is modified to replace the non-human (e.g. murine) framework sequences with human framework sequences, such that, generally, the only non-human sequences in the antibody are the CDR sequences.
  • Antibody humanisation is generally performed by a process known as CDR grafting, though any other technique in the art may be used. CDR grafting is well described in Williams, D.G. et al., Antibody Engineering Vol. 1 , edited by R. Kontermann and S. Dubel, Chapter 21 , pp. 319-339. In this process, humanisation of non-human variable domains involves intercalating the non-human CDRs from each immunoglobulin chain within the FRs of the most appropriate human variable region.
  • a chimeric antibody may be used in the methods provided herein.
  • a chimeric antibody is an antibody with variable domains derived from one species and constant domains derived from another.
  • a chimeric antibody for use herein may comprise non-human variable domains (e.g. mouse- or rat-derived variable domains) and human constant domains.
  • Chimeric antibodies may be generated using any suitable technique, e.g. recombinant DNA technology in which the DNA sequence of the variable domain (e.g. non-human variable domain) is fused to the DNA sequence of the constant domain (e.g. human constant domain) so as to encode a chimeric antibody.
  • a chimeric antibody fragment may be obtained either by using recombinant DNA technology to produce a DNA sequence encoding such a polypeptide, or by processing a chimeric antibody to produce the desired fragments, as described above.
  • the antibody or fragment thereof used herein may be an isolated antibody or isolated antigen-binding fragment of an antibody.
  • An "isolated antibody” or “isolated antigen-binding fragment of an antibody” as used herein means 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.
  • 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 exists or is naturally produced is an “isolated antibody” or an "isolated antigen-binding fragment of an antibody” for the purposes of the methods provided herein.
  • An isolated antibody or fragment thereof also includes an antibody or fragment thereof in situ within a recombinant cell.
  • Isolated antibodies or antigen-binding fragments thereof are antibodies or antigen-binding fragments thereof that have been subjected to at least one purification or isolation step.
  • An isolated antibody or antigenbinding fragment thereof may be substantially free of other cellular material and/or chemicals.
  • the antibody or fragment thereof for use herein may thus be synthesised by any method known in the art.
  • the antibody or fragment thereof may be synthesised using a protein expression system, such as a cellular expression system using prokaryotic (e.g. bacterial) host cells or eukaryotic (e.g. yeast, fungus, insect or mammalian) host cells. Cells which may be used in the production of the antibody or fragment thereof are discussed further below.
  • An alternative protein expression system is a cell-free, in vitro expression system, in which a nucleotide sequence encoding the specific binding molecule is transcribed into mRNA, and the mRNA translated into a protein, in vitro.
  • Cell-free expression system kits are widely available, and can be purchased from e.g. ThermoFisher Scientific (USA).
  • antibodies and fragments thereof may be chemically synthesised in a non-biological system. Liquid-phase synthesis or solid-phase synthesis may be used to generate polypeptides which may form or be comprised within the antibody or fragment thereof used herein. The skilled person can readily produce antibodies or fragments thereof using appropriate methodology common in the art.
  • the antibody or fragment thereof may be recombinantly expressed in mammalian cells, such as CHO cells.
  • mammalian cells such as CHO cells.
  • suitable mammalian cells for production of the antibody or fragment 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 a number of other cell lines including the mouse myeloma cell lines NSO and SP2/0.
  • the host cell when cultured under appropriate conditions, synthesises the antibody or antigen-binding fragment thereof for use herein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the antibody or fragment thereof may be isolated from synthesis, as discussed above.
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
  • the host cell line which produces the antibody or fragment thereof for use herein may stably express the antibody or fragment thereof or transiently express the antibody or fragment thereof.
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable regions of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • the antibodies for use in the methods provided herein may bind PAR2 on any suitable epitope, i.e. they may bind any epitope, so long as their binding to that epitope has the desired effect of inhibiting the activity of PAR2.
  • the antibody or fragment thereof for use in the present methods binds PAR2 at an epitope comprising the amino acid sequence set forth in SEQ ID NO: 11 . That is to say, the antibody or fragment 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 to 48 of full-length-PAR2.
  • SEQ ID NO: 11 is the epitope of MEDI0618, as determined by X-ray crystallography of a complex between a MEDI0618 Fab and an N-terminal PAR2 polypeptide (not shown in the Examples).
  • an antibody against PAR2 binds PAR2 at the epitope of SEQ ID NO: 11 can be determined by epitope mapping. Any method for epitope mapping known in the art may be used for this purpose. Examples of such methods include HDX, epitope excision, peptide panning, X-ray co-crystallography, 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 which specifically binds SEQ ID NO: 11 may also be specifically generated using the peptide of SEQ ID NO: 11 as the antigen, e.g. by immunising an animal (e.g. a mouse) with a peptide of SEQ ID NO: 11 or by using a peptide of SEQ ID NO: 11 as the affinity partner when generating antibodies by phage display.
  • an anti-PAR2 antibody or fragment thereof can be considered to recognise (i.e. specifically bind) the epitope of SEQ ID NO: 11 if the antibody or fragment thereof binds the native sequence of PAR2 (SEQ ID NO: 14) at the sequence set forth in SEQ ID NO: 11 . If the sequence of SEQ ID NO: 11 is mutated in PAR2, an antibody which specifically binds SEQ ID NO: 11 may nonetheless bind the mutated sequence, i.e. mutation of the epitope sequence of SEQ ID NO: 11 may be tolerated. However, an antibody which specifically binds the epitope of SEQ ID NO: 11 does not bind PAR2 at any other epitope, or at least recognises the epitope of SEQ ID NO: 11 more strongly than any other epitope on PAR2.
  • An antibody or fragment thereof can be considered to bind PAR2 at SEQ ID NO: 11 if it binds PAR2 at an epitope sequence within SEQ ID NO: 11 , even if the antibody or fragment thereof does not bind the entirety of the SEQ ID NO: 11 peptide.
  • an antibody or fragment thereof can be considered to bind PAR2 at SEQ ID NO: 11 if the epitope of the antibody or fragment includes SEQ ID NO: 11 (or part of SEQ ID NO: 11), but is not exclusively contained within SEQ ID NO: 11 .
  • an antibody or fragment thereof can be considered to bind SEQ ID NO: 11 if the antibody or fragment thereof binds an epitope which in part consists of SEQ ID NO: 11 (or part thereof) and in part consists of another sequence, e.g. sequence immediately N- or C-terminal to SEQ ID NO: 11 in PAR2.
  • the antibody or fragment thereof thus may bind 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 part of SEQ ID NO: 11 .
  • the antibody used in the methods provided herein may be advantageous for the antibody used in the methods provided herein to bind PAR2 with a higher affinity at physiological, extracellular pH (about pH 7.4) than at a slightly acidic pH, e.g. about pH 6.0.
  • Anti-PAR2 antibodies with such a binding characteristic have been found to display enhanced biological efficacy relative to anti-PAR2 antibodies whose binding to PAR2 is unimpacted by pH. Without being bound by theory, it is believed that this effect is because such pH-dependent target binding enables recycling of the antibody following PAR2 binding.
  • the extracellular domain of PAR2 is located extracellularly where the pH is about 7.4.
  • PAR2 Upon binding of an anti-PAR2 antibody to PAR2, PAR2 is internalised, whereupon it enters endosomal and lysosomal intracellular compartments, both of which are acidic (an endosome has a pH in the range of about 5.5-6).
  • an antibody binds PAR2 in a pH dependent manner, as described above, as the antibody-PAR2 complex is internalised in an endosome, as the endosomal pH drops the antibody will dissociate from PAR2. This allows the antibody to be recycled and released back out of the cell in the endocytic cycle, rather than degraded, increasing the serum halflife of the antibody and thus improving its pharmacokinetic profile in a therapeutic context.
  • An antibody may display pH-dependent recognition of PAR2 if it comprises at least 1 histidine residue in its CDRs.
  • the histidine side chain has a pK a of about 6.0, so below this pH level the imidazole ring of the histidine side chain is generally protonated (and thus neutral), whereas above this pH level the imidazole ring is generally unprotonated (and thus positively charged).
  • This change in charge of the histidine side chain can substantially alter the affinity for its target of an antibody which contains one or more histidine residues in its CDRs.
  • An antibody or fragment thereof for use herein may comprise a histidine in VLCDR1 , VLCDR2, VLCDR3, VHCDR1 , VHCDR2 and/or VHCDR3.
  • An antibody or fragment thereof for use herein may in total comprise at least 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more histidine residues in its CDR sequences. Such histidine residues may be natively present in the CDR sequences or may be introduced by mutation (e.g. site directed mutagenesis). A histidine residue may be present at any location within the CDR sequences.
  • the antibody for use herein may bind PAR2 at pHs of 7.4 and 6.0 with affinities as discussed above. For instance, the antibody for use herein may bind PAR2 with at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater affinity at pH 7.4 than at pH 6.0.
  • the antibody or antigen-binding fragment thereof for use in the methods provided herein may in particular comprise CDRs as follows:
  • VLCDR1 comprising the sequence set forth in SEQ ID NO: 1 ;
  • VLCDR2 comprising the sequence set forth in SEQ ID NO: 2;
  • VLCDR3 comprising the sequence set forth in SEQ ID NO: 3;
  • VHCDR1 comprising the sequence set forth in SEQ ID NO: 4;
  • VHCDR2 comprising the sequence set forth in SEQ ID NO: 5; and VHCDR3 comprising the sequence set forth in SEQ ID NO: 6.
  • the CDRs may consist of SEQ ID NOs: 1-6, respectively, as set out above.
  • the antibody or antigen-binding fragment thereof for use herein may comprise a heavy chain variable domain ( H) 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 thereto.
  • H heavy chain variable domain
  • the antibody or antigen-binding fragment thereof for use herein may comprise a light chain variable domain (V L ) 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 thereto.
  • V L light chain variable domain
  • the antibody or antigen-binding fragment thereof for use herein may comprise:
  • V H a heavy chain variable domain 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 thereto;
  • a light chain variable domain 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 thereto.
  • the antibody or antigen-binding fragment thereof comprises a V H with at least 70 % identity to SEQ ID NO: 7, but less than 100 % identity to SEQ ID NO: 7, this may be subject to the proviso that the CDRs are as defined above, i.e. that 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 to say, when the heavy chain variable region comprises a variant of SEQ ID NO: 7, generally all variation in the heavy chain variable domain sequence relative to SEQ ID NO: 7 is found within the framework regions.
  • the antibody or antigen-binding fragment thereof comprises a L with at least 70 % identity to SEQ ID NO: 8, but less than 100 % identity to SEQ ID NO: 8, this may be subject to the proviso that the CDRs are as defined above, i.e. that 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 to say, when the light chain variable domain comprises a variant of SEQ ID NO: 8, generally all variation in the light chain variable domain sequence relative to SEQ ID NO: 8 is found within the framework regions.
  • the 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 thereto.
  • the 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 thereto.
  • the antibody 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 thereto;
  • 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 thereto.
  • the antibody comprises a heavy chain which is a variant of SEQ ID NO: 9
  • this may be subject to the proviso that the CDRs are as defined above, i.e. that 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 to say, when the heavy chain comprises a variant of SEQ ID NO: 9, generally all variation in the heavy chain sequence relative to SEQ ID NO: 9 is found within the constant domain and the framework regions of the variable domain.
  • the antibody comprises a light chain which is a variant of SEQ ID NO: 10
  • this may be subject to the proviso that the CDRs are as defined above, i.e. that VLCDR1 comprises the amino acid sequence of SEQ ID NO: 1
  • VLCDR2 comprises the amino acid sequence of SEQ ID NO: 2
  • VLCDR3 comprises the amino acid sequence of SEQ ID NO: 3. That is to say, when the light chain comprises a variant of SEQ ID NO: 10, generally all variation in the light chain sequence relative to SEQ ID NO: 10 is found within the constant domain and the framework regions of the variable domain.
  • the antibody with the heavy chain amino acid sequence set forth in SEQ ID NO: 9 and the light chain amino acid sequence SEQ ID NO: 10 is referred to herein as MEDI0618.
  • Sequence identity of variants of the sequences set out above may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programmes that make pairwise or multiple alignments of sequences are useful, for instance EMBOSS Needle or EMBOSS stretcher (both Rice, P. et al., Trends Genet. 16, (6) pp. 276-277, 2000) may be used for pairwise sequence alignments while Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 201 1 ) or MUSCLE (Edgar, R.C., Nucleic Acids Res. 32(5): 1792-1797, 2004) may be used for multiple sequence alignments, though any other appropriate programme may be used. Whether the alignment is pairwise or multiple, it must be performed globally (i.e. across the entirety of the reference sequence) rather than locally.
  • Sequence alignments and % identity calculations may be determined using for instance standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1.
  • the standard EMBOSS Needle parameters may be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may alternatively be used.
  • Variants of the sequences set out herein i.e. sequences with 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 relative to the original sequences.
  • 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 thus a conservative substitution of an amino acid important for the structure or function of a polypeptide may be expected to affect polypeptide structure/function less than a non-conservative amino acid substitution at the same position. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.
  • polar side chains e.g. asparagine, glutamine, serine, threonine, tyrosine
  • non-polar side chains e.g. glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine.
  • a conservative amino acid substitution may be considered to be a substitution in which a particular amino acid residue is substituted for a different amino acid in the same family.
  • a substitution of an amino acid residue may equally be a non-conservative substitution, in which one amino acid is substituted for another with a side-chain belonging to a different family.
  • an antibody or fragment thereof which comprises a variant of the MEDI0618 variable domain sequences (i.e. a variable domain which is a variant of SEQ ID NO: 7 or SEQ ID NO: 8, as set out above) or full chain sequences (i.e. a heavy or light chain which is a variant of SEQ ID NO: 9 or SEQ ID NO: 10), the variant may have equivalent activity to MEDI0618 or a corresponding fragment thereof.
  • an antibody which is a variant of MEDI0618 i.e.
  • an antibody comprising a variant sequence as described above may bind PAR2 with an affinity which is equivalent to MEDI0618, which is no lower than the affinity with which MEDI0618 binds PAR2, or which is not substantially lower than the affinity with which MEDI0618 binds PAR2.
  • a variant of MEDI0618 may bind PAR2 with a KD as set out above.
  • a variant of MEDI0618 may be considered to bind PAR2 with an affinity which is not substantially lower than the affinity with which MEDI0618 binds PAR2 if the variant of MEDI0618 binds PAR2 with an affinity which is reduced by no more than 5 %, 10 %, 15 %, 20 % or 25 % compared to that of MEDI0618.
  • An antibody which is a variant of MEDI0618 may display other biological activities which are equivalent to those displayed by MEDI0618, e.g. in respect of PAR2 inhibition (as measured by a calcium flux assay as described above), PAR2 internalisation and any other effector functions displayed by MEDI0618.
  • the anti-PAR2 antibodies used in the methods provided herein may be IgG antibodies comprising a constant domain (Fc domain) comprising one or more mutations relative to the native Fc sequence which enhance or diminish antibody binding to the FcRn receptor, e.g. at acidic pH as compared to neutral pH.
  • the anti-PAR2 antibodies used herein may comprise a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g. in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in 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.
  • the position number means the position of the amino acid in a full-length IgG heavy chain (i.e. position 1 is defined as the first amino acid of the variable domain, excluding the signal sequence).
  • the position number means the position of the amino acid in a full-length human IgG heavy chain, in particular a full-length human IgG 1 heavy chain.
  • the listed amino acids are the residues which may be present at the listed positions in the modified chains (i.e. are amino acids with which the native amino acids may be substituted).
  • the Fc domain may comprise: 428L (e.g. M428L) and 434S (e.g. N434S) modifications; 428L, 259I (e.g. V259I) and 308F (e.g. V308F) modifications; 433K (e.g. H433K) and 434 (e.g. 434Y) modifications; 252, 254, and 256 (e.g.
  • 428L e.g. M428L
  • 434S e.g. N434S
  • 433K e.g. H433K
  • 434 e.g. 434Y
  • the Fc domain may comprise any combination of the aforementioned modifications.
  • the antibody may comprise the triple mutation L234F/L235E/P331S ("TM") in its Fc domain.
  • TM causes a profound decrease in the binding activity of human IgG 1 molecules to human Fey receptors including FcyR1 (CD64), FcyR2A (CD32A) and CD16 (FcyRIII), as detailed in e.g., Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 64:700- 704 (2008).
  • Antibodies with increased half-lives may also be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor.
  • the introduction of the triple mutation M252Y/S254T/T256E ('YTE') into the C H 2 domain of human IgG molecules causes an increase in their binding to the human neonatal Fc receptor (FcRn), as described in U.S. Patent No. 7.083,784, the contents of which are herein incorporated by reference in their entirety.
  • the antibodies used herein may comprise the YTE modifications.
  • the antibody or fragment thereof used in the present methods may be an antibody or antibody fragment which has a different sequence to MEDI0618 (e.g. has different CDRs to MEDI0618, or different variable domain framework region sequences) but which is bioequivalent to MEDI0618 (or bioequivalent to the corresponding fragment of MEDI0618).
  • Two antibodies or antigen-binding fragments thereof are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single doses or multiple doses.
  • Some antibodies or antigen-binding fragments will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • Two antibodies or antigen-binding fragments thereof may be considered bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.
  • two antibodies or antigen-binding fragments thereof may be considered bioequivalent if a patient can be switched one or more times between the two products without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antibodies or antigen-binding fragments thereof may be considered bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • an antibody which is bioequivalent to MEDI0618 may bind the same epitope as MEDI0618 (i.e. SEQ ID NO: 11) with a similar affinity and thereby cause a similar degree of inhibition of PAR2 activity as MEDI0618 (as measured by e.g. calcium flux assay, as described above.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.
  • the antibody or antigen-binding fragment thereof may be administered to the subject within a pharmaceutical composition comprising at least one pharmaceutically acceptable diluent, carrier or excipient.
  • pharmaceutically acceptable refers to ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient.
  • the nature of the composition and carriers or excipient materials, dosages etc. may be selected in routine manner according to choice and the desired route of administration, etc. Dosages may likewise be determined in routine manner and may depend upon the nature of the molecule, age of patient, mode of administration etc.
  • compositions for use herein include liquid solutions or syrups, solid compositions such as powders, granules, tablets or capsules, creams, ointments and any other style of composition commonly used in the art.
  • suitable pharmaceutically acceptable diluents, carriers and excipients for use in such compositions are well known in the art.
  • suitable excipients include lactose, maize starch or derivatives thereof, stearic acid or salts thereof, 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, talcum, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, gelatin, oil, alcohol, detergents and emulsifiers such as the polysorbates. Stabilising agents, wetting agents, emulsifiers, sweeteners etc. may also be used.
  • Liquid pharmaceutical compositions may include one or more of the following: sterile diluents such as water for injection, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as dextrose.
  • a parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • the mode of administration of the antibody or antigen-binding fragment thereof for use herein may be selected by the skilled physician.
  • an appropriate dosage may be determined by the skilled person taking into account the age and size of the patient, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • Effective dosages and schedules for administering anti-PAR2 antibodies or antigen-binding fragments thereof may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Interspecies scaling of dosages can be performed using well- known methods in the art (see e.g. Mordenti et al., 1991 , Pharmaceut. Res. 8:1351).
  • aCGRP-IR Inadequate Responder to anti-CGRP therapy
  • CGRP calcitonin gene-related peptide
  • the injectors were connected to Tygon tubing (Cole Parmer Co. Vernon Hills, Illinois, USA) which was attached to a 25 mL Hamilton syringe (Hamilton, Reno, Nevada, USA). The injector was inserted through the sagittal and lambdoid suture junction region and the injection was delivered onto the dura mater. The total volume injected was 5 mL and each injector was used a maximum of four times.
  • mice received s.c. MEDI0618 IgGITM at 50 mg/kg or isotype control (NIP228 IgGITM,; cone 10.7 mg/ml) at 50 mg/kg. All mice received supradural administration of inflammatory mediator (IM).
  • IM inflammatory mediator
  • mice received 3 days of von Frey (VF) acclimation.
  • Baseline 1 (BL1) measurement was performed followed by antibody treatment 24 hrs prior to the IM time course.
  • IM time course mice received VF acclimation for 2 hours followed by BL2 measurement.
  • IM supradural administration was performed and then VF measurements were taken 30 min, 1 , 2, 3, 4, and 5 hours after supradural IM administration.
  • the IM mix comprised 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.
  • mice received 3 days of VF acclimation for 2 hours each.
  • the animals’ heads were shaved 1 day before the time course/BL1/I.P. treatment with olcegepant at 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), 6 animals per group.
  • 15 min after treatment started BL2 was measured, after which all mice received supradural injection of CGRP.
  • 1 pg CGRP was administered in a volume of 5 pL, using the 0.7 mm injector.
  • VF evaluation was performed 5, 10, 20, 40, 60, 120 and 180 min after injection. Shaving and supradural administration were performed under isofluorane light anaesthesia.
  • mice received 3 days of VF acclimation for 2 hours each.
  • the animals’ heads were shaved 1 day before the time course/BL1/I.P. treatment with olcegepant at 1 mg/kg/10 mL or vehicle (20 % DMSO/80 % saline), 6 animals per group.
  • 15 min after treatment started BL2 was measured, after which all mice received supradural injection of 5 pL IM (as defined above) using the 0.7 mm injector.
  • VF evaluations were performed 30 min, 1 , 2, 3, 4 and 5 h after injection. Shaving and supradural administration were performed under isofluorane light anaesthesia.
  • mice received s.c. MEDI0618 IgGITM (Lot SP16-112) at 50 mg/kg; 4 animals received s.c. isotype control (NIP228 IgGITM, Lot SP14-302; cone 10.7 mg/ml) at 50 mg/kg. All mice received supradural administration of IM. Protocol: mice received 3 days of VF acclimation. The animals’ heads were shaved on day 3, after which BL1 was measured followed by MEDI0618 treatment 24 hrs prior to the IM time course. In the IM time course mice received VF acclimation for 2 hours followed by BL2 measurement. IM supradural administration was then performed and VF measurements taken 30 min, 1 , 2, 3, 4, and 5 hours after supradural IM administration.
  • Trigeminal neurons were harvested from 6-8 week old female C57BL6 mice via methods previously described (Malin et al., 2007 Nature Methods, DOI: 10.1038/nprot.2006.461). Briefly, following euthanasia and decapitation, the overlying skin, skull and brain were removed to reveal the trigeminal ganglion in the base of the skull. The trigeminal ganglion was dissected and chopped before undergoing sequential digestion in papain, followed by collagenase type II and dispase type II. Neurons were separated from myelin and nerve debris via centrifugation through a Percoll gradient (12.5 % over 28 % Percoll).
  • Cells were diluted to high density by adding 30 pl media (L15 + 5 % Fetal Bovine Serum (FBS) + 2 % 1 M HEPES + 1 % penicillin/streptomycin) per trigeminal pair. 40 pl volumes of cell mix were spotted onto 12 mm cover glass pre-coated with poly-D-lysine (PDL) and laminin (Biocoat #1 German Glass) placed in 24 well plates. Wells were flooded after 30 minutes cell attachment and maintained in F12 (Ham’s) Nutrient Mix + 10 % FBS + 1 % penicillin/streptomycin.
  • PDL poly-D-lysine
  • laminin Biocoat #1 German Glass
  • Trigeminal cultures were incubated at 37°C and 5 % CO 2 for 2-4 days in vitro before calcium imaging experiments.
  • cytosolic calcium concentration were undertaken to measure functional activity of CGRP receptor and PAR2 activation following treatment with specific agonists: rat CGRP peptide (CGRP; Tocris) and 2-Furoyl-LIGRLO-amide (LIGRLO; Peptides International) respectively.
  • Neuronal activity was identified from non-neuronal activity via responsivity to 20 mM KCI.
  • Mouse trigeminal cultures were loaded with 5 pM Fura-2 ratiometric calcium dye in Hank's Balanced Salt Solution +/+ (HBSS) for 30 minutes at 37°C and 5 % CO 2 before transfer to a perfusion chamber (Warner Instruments) containing HBSS.
  • HBSS Hank's Balanced Salt Solution +/+
  • Widefield calcium imaging was performed with an 1X81 inverted microscope (Olympus) equipped with a sCMOS Orca R2 camera (Hamamatsu). Time series were recorded with a frame rate of 400 ms and using sequential 340 nm (+/-25 nm) and 387 nm (+/-11 nm) excitation and 510 nm (+/-40 nm) bandpass emission capture. Cultures were perfused at a flow rate of 1 .1 ml/minute with HBSS +/- treatments via a gravity flow perfusion system equipped with VC-6 valve controller (Warner Instruments) to switch treatment lines.
  • VC-6 valve controller Warner Instruments
  • CGRP and LIGRLO were sequentially treated with 1 pM CGRP (40 seconds), HBSS washout (100 seconds), 10 pM LIGRLO (40 seconds), HBSS washout (100 seconds), 20 mM KCI (30 seconds) and finally HBSS washout (80 seconds).
  • ROIs Regions of interest (ROIs) were selected for putative neurons and ratios of fluorescence intensity (387 nm over 340 nm excitation) were calculated for each timepoint, for each ROI.
  • Bespoke Matlab routines were applied for trace quantification. First, traces with unstable baselines and traces belonging to non-neuronal cells were removed. Next, cells with activity within CGRP and LIGRLO treatment periods were identified (ratio >10 % over baseline), and the maximum amplitude within these periods calculated.
  • Human dural fibroblasts (Innoprot catalogue number P10375) were used between P3 & P7.
  • Primary human dural microvascular endothelial cells (Creative Biolabs; cat. No. NCL-21 P6-022) were used between P1-3.
  • Cells were harvested from a T175 flask at 50 % confluence by treatment with accutase for 5 mins, spun down at 1000 rpm for 5 mins then resuspended in culture medium.
  • Human dural fibroblasts culture media consisted ofFibroblast basal media, 500 ml; Foetal bovine serum, 10 ml; fibroblast growth supplement, 100X, 5 ml; Penicillin/Streptomycin, 5 ml.
  • Human dural microvascular endothelial cell culture media consisted of were Endothelial Growth Media 2 (EGM-2) plus commercial supplements (Lonza). Cells were diluted to 333,333 cells/ml and plated at 5 x 10 3 cells per well (30 pl per well) in Cell Coat Poly-D-Lysine-coated 384 well plates (black, uclear, #781946, Lot#E11020JH, Greiner). Cells were then placed in a humidified incubator at 37°C for 24 hrs prior to commencing the experiment.
  • Drugs were made up in assay buffer and added sequentially to the cell plate (10 pl per addition).
  • Assay buffer For determination of antibody (lgG1) potency, cells were pre-treated with IgGs diluted in assay buffer for up to 1 h at room temperature prior to addition of the PAR2 agonist 30nM matriptase.
  • Rat pharmacokinetic studies were performed in male Sprague Dawley rats (200-215 g) at an intravenous dose level of 1 mg/kg.
  • a Gyrolab Bioaffy 200 CD containing a column pre-packed with streptavidin-coated beads, was functionalised with a biotinylated capture antibody (mouse anti-human IgG (CH2 domain; R10Z8E9)).
  • a biotinylated capture antibody mouse anti-human IgG (CH2 domain; R10Z8E9).
  • Standards and samples were passed through the column and the capture antibody bound monoclonal antibody drug in the serum.
  • a fluorophore-labeled detection antibody was then passed through the column to bind to the captured analyte.
  • the concentration of detection reagent bound to the analyte is calculated from the fluorescence measured by the fluorescence detector in the Gyrolab Workstation.
  • pharmacokinetic parameters were estimated using Phoenix WinNonlin pharmacokinetic software version 1.4 (Pharsight, USA) using a non-compartmental approach consistent with the intravenous infusion route of administration. All parameters were generated from individual concentrations in serum and estimated using sampling times relative to the start of each dose administration (within an acceptable tolerance limit).
  • the area under the serum concentration versus time curve (AUC) was calculated using the log up, linear down trapezoidal method with linear interpolation.
  • the terminal elimination phase of each concentration versus time curve was identified using at least the final three observed concentration values. The slope of the terminal elimination phase was determined using log linear regression on the unweighted concentration data.
  • a mouse model of migraine-like pain was used to investigate the therapeutic effect of the anti-PAR2 antibody MEDI0618 for this indication. It is known that migraine pain can be mediated by CGRP and so administration of a CGRP inhibitor is expected to prevent the development of migraine-like pain in a mouse model.
  • the site of action of PAR2 in migraine could include non-neuronal cells of the trigeminovascular system such as dural fibroblasts and dural microvascular endothelial cells.
  • MEDI0618 showed improved potency over PAR650097 when measuring the ability to inhibit matriptase- stimulated calcium influx in human dural fibroblast cells (FIG. 4A).
  • MEDI0618 is first described in WO 2018/167322, where it is designated PaB670129.
  • WO 2018/167322 describes the generation of MEDI0618 from the parent antibody Par0067.
  • several variants of Par0067 are generated by mutation of various residues in VHCDR2, VHCDR3 and/or VLCDR3 of Par0067 to histidine, in order to generate antibodies which display pH- dependent binding. As set out above, this is advantageous in therapeutic anti-PAR2 antibodies.
  • the MEDI0618 CDRs comprise multiple histidine residues, all of which were introduced by mutation of the Par0067 CDR sequences. Serum clearance of MEDI0618 was compared in rats to clearance of the parent antibody Par0067, and various other different Par0067 derivatives containing histidine substitutions in their CDRs.
  • MEDI0618 shows decreased serum clearance compared to the parent antibody (referred to as P67) and superior (i.e. slower) serum clearance (CL) than any of the other Par0067-derived antibodies tested. This is also set out in the table below.

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Abstract

L'invention concerne de manière générale des compositions et des procédés qui sont utiles dans le traitement de la migraine. Plus spécifiquement, les procédés et les compositions peuvent être utilisés pour traiter, soulager ou prévenir la migraine chez des sujets qui sont des répondants inadéquats à une thérapie anti-5 CGRP (aCGRP-IR) ou chez des sujets qui ne réagissent pas à une thérapie anti-CGRP (peptide lié au gène de la calcitonine) ou à un inhibiteur du peptide lié au gène de la calcitonine (CGRP).
PCT/EP2024/051579 2023-01-25 2024-01-24 Thérapie contre la migraine Ceased WO2024156720A1 (fr)

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CN202480014318.7A CN120835790A (zh) 2023-01-25 2024-01-24 偏头痛疗法
EP24702274.2A EP4655068A1 (fr) 2023-01-25 2024-01-24 Thérapie contre la migraine
AU2024212070A AU2024212070A1 (en) 2023-01-25 2024-01-24 Migraine therapy
MX2025008572A MX2025008572A (es) 2023-01-25 2025-07-23 Terapia contra la migraña

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
WO2018167322A1 (fr) 2017-03-16 2018-09-20 Medimmune Limited Anticorps anti-par2 et leurs utilisations

Patent Citations (2)

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US7083784B2 (en) 2000-12-12 2006-08-01 Medimmune, Inc. Molecules with extended half-lives, compositions and uses thereof
WO2018167322A1 (fr) 2017-03-16 2018-09-20 Medimmune Limited Anticorps anti-par2 et leurs utilisations

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