CN121358766A - Combination therapy - Google Patents

Abstract

The present invention relates to compositions comprising eplerenone and LuAG09222. The composition can be used for treating or preventing, for example, headache, pain, migraine, cluster headache, or endometriosis.

Description

Combination therapy

Background

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/509,854, filed on day 23, 6, 2023, the contents of which are incorporated by reference in their entirety.

Sequence Listing disclosure

The contents of the electronic sequence listing (1143257 o014013.Xml; size: 25,446 bytes; and date of creation: 2024, 6, 17 days) are incorporated herein by reference in their entirety.

For reasons of sequence length, it is not allowed to enter sequences in St.26 Xml files

Table A below lists the sequences present in priority application U.S. provisional application No. 63/509,854 (identified above, which is incorporated herein by reference in its entirety), but for reasons of sequence length, these sequences cannot be included in the 1143257o014013.xml file filed herewith.

Technical Field

The present invention relates to a composition comprising two antibodies or fragments thereof (including Fab fragments) specific for different targets. One of these antibodies or antibody fragments specifically binds to a human calcitonin gene-related peptide (hereinafter referred to as "CGRP"), and the other antibody or antigen-binding fragment thereof specifically binds to pituitary adenylate cyclase activating polypeptide ("PACAP").

Background

Calcitonin Gene Related Peptide (CGRP) was produced as a multifunctional neuropeptide of 37 amino acids in length. Both forms of CGRP, namely CGRP-alpha and CGRP-beta, are present in humans and have similar activity. CGRP-alpha and CGRP-beta have three amino acid differences in humans and are derived from different genes. CGRP is released from many tissues (such as the trigeminal nerve) that, upon activation, release meningoendopeptides, mediating neurogenic inflammation characterized by vasodilation, vascular leakage, and mast cell degradation. Durham, P.L., new Eng.J. Med. [ J.New England medical journal ], 350 (11): 1073-75 (2004). The biological effects of CGRP are mediated through the CGRP receptor (CGRP-R), which consists of seven transmembrane components and a receptor-associated membrane protein (RAMP). CGRP-R also requires the activity of the Receptor Component Protein (RCP), which is critical for efficient coupling of G protein to adenylate cyclase and production of cAMP. Doods, H., curr. Op. invest. Drugs [ current opinion of research Drugs ], 2 (9): 1261-68 (2001).

Several antibodies have been approved for the treatment of migraine. These antibodies bind CGRP or CGRP receptors. FDA approved antibodies include Aimovig (eplerenone You Shan antibody), ajovi (rimainab), emgality (ganaxainab) and Vyepti (eplercanizumab). The present invention relates to Vyepti (eplerizumab or Ab6 as used herein), which is a drug approved for the prophylactic treatment of migraine. The recommended dose is 100 mg for every 3 months of intravenous infusion. Some patients may benefit from a dose of 300 mg administered every 3 months.

Pituitary adenylate cyclase activating polypeptide ("PACAP") is a member of the secretin/vasoactive intestinal peptide ("VIP")/growth hormone releasing hormone ("GHRH") family. PACAP is a multifunctional vasodilating peptide that exists in two alpha-amidated active forms, one with 38 amino acids (PACAP 38) and the other with 27 amino acids (PACAP 27). Both peptides have the same N-terminal 27 amino acids and are synthesized from the same precursor protein preproPACAP (see, moody et al, curr. Opin. Endocrinol. Diabetes Obes. [ recent views of endocrinology, diabetes and obesity ], 18 (1): 61-67, 2011). PACAP38, as the more ubiquitous active form, accounts for up to 90% of the PACAP forms in mammalian tissues (see, kaiser and Russo, neuropeptides [ neuropeptides ], 47:451-461, 2013). The sequence of PACAP38 is identical in all mammals and differs from the ortholog of birds and amphibians by only one amino acid (see Vaudry et al, pharmacol. Rev. [ pharmacological review ], 52:269-324, 2000). The secretin/VIP/GHRH family includes the mammalian peptides histidine methionine ("PHM"), secretin, glucagon-like peptide-1 ("GLP 1"), glucagon-like peptide-2 ("GLP 2"), glucose-dependent insulinotropic polypeptide ("GIP"), and growth hormone releasing factor ("GRF"). PACAP27 has 68% sequence identity at the amino acid level to VIP (see, vaudry et al, 2000).

The biological effects of PACAP are mediated via three different G-protein coupled receptors, PAC1-R, vasoactive intestinal peptide receptor type 1 ("VPAC 1-R"), and vasoactive intestinal peptide receptor type 2 ("VPAC 2-R"). These receptors are expressed in different tissues. PAC1-R is particularly abundant in the nervous system (e.g., olfactory bulb, thalamus, hypothalamus, cerebellum, and dorsal horn of spinal cord), pituitary and adrenal glands. In contrast, VPAC1-R and VPAC2-R are expressed primarily in the lung, liver and testes, although both receptors have also been detected in other tissues. VPAC1-R expression was detected in the nervous system (e.g., cerebral cortex and hippocampus), smooth muscle cells, megakaryocytes and platelets of the lung, liver, intestine. VPAC1-R binds to receptor-associated membrane proteins ("RAMP", in particular RAMP 2) (see Christopoulos et al, J. Biol. Chem. [ J. Biol., 278:3293-3297, 2002). The VPAC2-R expression profile includes the nervous system (e.g., thalamus, hippocampus, brain stem and dorsal root ganglion ("DRG")), cardiovascular system, gastrointestinal system, pancreas and reproductive system (see Usdin et al, endocrin. J. Endocrine, 135:2662-2680, 1994; shewand et al, neurosci. J. Neuroscience, 67:409-418, 1995).

PACAP is presumed to play a role in a variety of diseases and disorders including, but not limited to, migraine, headache, and pain. Migraine is thought to be associated with neurovascular factors. About 10% of american adults suffer from migraine and migraine is typically accompanied by severe headache. About 20% -30% of migraine sufferers experience aura, including focal neurological phenomena prior to and/or accompanying the event. Several observations demonstrate the role of PACAP in migraine (1) in the course of a human migraine attack (burst), the plasma levels of PACAP are elevated compared to the inter-attack levels (see Tuka et al, CEPHALALGIA [ headache ], 33 (13): 1085-1095, 2013); (2) infusion of PACAP38 causes headache in healthy subjects and causes headache in migraine patients, followed by migraine-like attacks (see Schytz et al, brain [ Brain ], 132:16-25, 2009; and Amin et al, brain [ Brain ], 137:779-794, 2014); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see Kaiser and Russo, neuropeptides [ neuropeptides ], 47:451-461, 2013); (4) PACAP-induced migraine is associated with photophobia, nausea, and is responsive to the triptans (see, amin et al [ Brain ], 2012-149). PACAP has also been shown to induce vasodilation, photophobia, and mast cell degranulation and neuronal activation (see Markovics et al, neurobiology of Disease [ neurobiology of disease ], 45:633-644, 2012; baun et al, CEPHALALGIA [ headache ], 32 (4): 337-345, 2012; chan et al, pharmacology & Therapeutics [ Pharmacology and Therapeutics ], 129:332-351, 2011).

PACAP may also be involved in diseases and disorders other than migraine, headache and pain. For example, PACAP may be associated with anxiety disorders (WO 2012/106407), thrombocytopenia (WO 2004/062684) and inflammatory skin diseases (WO 2010/007175) or may even play a causal role in these diseases. PACAP and PAC1-R polymorphisms are associated with post-traumatic stress syndrome ("PTSD"), major depressive disorder, and generalized anxiety disorder in women, suggesting a role for PACAP in these disorders. In addition, supporting the role of PACAP in thrombocytopenia, patients with trisomy 18 have excess PACAP and exhibit defective megakaryocyte maturation (see Schytz et al, 2010; and Moody et al, curr. Opin. Endocrinol. Diabetes Obes. [ recent views of endocrinology, diabetes and obesity ], 18 (1): 61-67, 2011).

In addition, PACAP and other neuropeptides such as calcitonin gene-related peptide ("CGRP"), substance P, neurokinin A, bradykinin and endothelin-1 are expressed in the lower urinary tract ("LUT") (see Arms and Vizzard, handbook exp. Pharmacol. [ Handbook of Experimental pharmacology ], 202:395-423, 2011), and are reported to play a role in LUT dysfunction and urinary tract disorders such as urinary tract infection ("UTI"), abnormal excretion, urgency, nocturia, urinary incontinence, overactive bladder and pain associated with such conditions.

PACAP and PACAP receptors are also believed to regulate inflammatory and neuropathic Pain and are associated with pro-nociception and antinociception (see Davis-Taber et al, J. Pain journal, 9 (5): 449-56, 2008). PACAP is also reported to be required for spinal cord desensitization and neuropathic pain induction (see Mabuchi et al J. Neurosci. [ journal of neuroscience ], 24 (33): 7283-91, 2004). In addition, morphine withdrawal behavior has been reported to be altered in PACAP receptor deficient mice, further indicating the role of PACAP in morphine withdrawal anxiolytic response (see, martin et al, mol. Brain Res. [ molecular Brain research ], 110 (1): 109-18, 2003).

The present invention relates to anti-PACAP antibodies (all aliases of the same antibody) designated as LuAG09222, ab10.H3 or ALD1910 (Moldovan Loomis et al, J Pharmacol Exp Ther [ journal of pharmacology and experimental therapeutics ] 369:26-36, month 4 of 2019). The drug was tested in various clinical trials of migraine at different doses and showed significant results in just the proof of concept study of migraine (clinicaltrias. Gov No. NCT05133323; and THE PHARMA LETTER [ pharmaceutical communication ] 2023, 4 th month, 20 day "Lundbeck posts positive Phase IIa results with LuAG09222 [ the positive IIa result of LuAG09222 published by northbound corporation ]").

The present invention relates to the use of eplerenone and LuAG09222 as combination therapies or preferably in a composition comprising both drugs. The combination therapy or composition may be used to treat a medical condition, optionally headache, episodic or chronic migraine, cluster headache, endometriosis or pain.

Disclosure of Invention

The present disclosure provides methods of treating headache in a patient comprising administering to a patient in need thereof an effective amount of an anti-CGRP antibody (e.g., eplerizumab) or antibody fragment thereof as disclosed herein and an effective amount of an anti-PACAP antibody (e.g., luAG 09222) or antibody fragment thereof.

According to one embodiment, the present invention relates to the treatment of migraine. The antibody treatment may be initiated at an inter-seizure period (i.e., between migraine attacks) or during a burst period (i.e., during a migraine attack). The migraine may include, for example, chronic migraine or episodic migraine, and in particular aspects of the invention, the patient is suffering from chronic migraine. In the present invention, the anti-CGRP antibody is denoted Ab6, eplerenone or Vyepti, which are all intended to refer to the same antibody. The anti-PACAP antibody is denoted LuAG09222, ALD1910, or ab10.h3, all of which are intended to refer to the same antibody.

The antibodies of the invention may be produced in yeast or mammalian cells, such as pichia pastoris or CHO cells. The antibodies of the invention may be in a composition comprising histidine (10-50 mM), polysorbate 80 or poloxamer 188 (0.005-0.05% w/v) at a pH of 5.0-6.8. The composition may additionally comprise tonicity agents, optionally NaCl (10-150 mM), sorbitol (50-250 mM) and/or arginine (50-250 mM). The total amount of the two antibodies combined may be 150 mg/ml, e.g. such that eplerizumab is present at 100 mg/ml and LuAG09222 is present at 50 mg/ml. The formulation may be administered to the patient subcutaneously or intravenously (iv). Further embodiments of the formulations and co-formulations are described herein and in the claims. As used in the present invention, histidine also includes the L-form of histidine (L-histidine), sorbitol includes the L-sorbitol form, and arginine includes the L-arginine form.

In another embodiment of the invention, the combination therapy or composition may be used in a method for reducing, treating or preventing migraine (with or without aura), cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, weight loss, pain, hemiplegic migraine, cluster headache, migraine neuropathic pain, chronic headache, tension headache, general headache, hot flushes, chronic onset migraine, secondary headache caused by potential structural problems of the head or neck, cerebral neuralgia, sinus headache (e.g., like sinusitis related headache) or allergy-induced headache or migraine. The antibodies and antibody fragments of the invention are particularly useful in the treatment, prevention, amelioration, management or reduction of risk of one or more of overactive bladder and other urinary disorders including bladder infection, pain, chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, ocular pain, dental pain, postoperative pain, wound-related pain, burn-related pain, diabetes, non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders, inflammation, arthritis, bronchial hyperreactivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, brain trauma, epilepsy, neurodegenerative diseases, dermatological disorders including pruritus, neuropathic skin redness, skin roses and erythema, inflammatory bowel disease, irritable bowel syndrome, cystitis, dysmenorrhea and other disorders that may be treated or prevented or conditions ameliorated by antagonizing CGRP and/or PACAP signaling. Of particular importance are acute or prophylactic treatments of headache, including migraine and cluster headache, as well as other pain-associated disorders and endometriosis.

In another embodiment of the invention, the combination therapy or composition may be used in a method for reducing, treating or preventing gastroesophageal reflux and/or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menopause, childbirth, menopause, prostatitis or pancreatitis.

In migraine, the hyperactivation of the trigeminal nerve results in increased release of calcitonin gene-related peptide (CGRP) and other peptides, thereby causing the release of neurogenic pro-inflammatory mediators. Over time, these mediators further increase CGRP synthesis and release, corresponding to typical migraine attacks. CGRP mediates its actions via vasodilation of cerebral and arterioles, activating adenylate cyclase in smooth muscle cells (mainly in the trigeminal vascular network), which in turn leads to abnormal activation of nociceptors and the induction of pain.

Based on several lines of evidence, pituitary Adenylate Cyclase Activating Polypeptides (PACAP) are thought to contribute to the pathophysiology of headache, and studies have demonstrated elevated PACAP levels in bursty phase compared to inter-seizure phase in migraine patients. In addition, PACAP may be released during migraine attacks, and PACAP levels in chronic migraine sufferers are elevated.

Additional evidence comes from a challenge study in which the administration of both PACAP-38 and PACAP-27 resulted in migraine-like events in both healthy subjects and migraine patients. PACAP may also play a role in cluster headache. Similarly, studies have demonstrated that PACAP levels are also elevated during clustered event bursts as compared to inter-seizure episodes.

CGRP and PACAP neuropeptides have overlapping positions and physiological similarities, but also show different biological effects.

This suggests that dual neutralization of CGRP and PACAP peptides will produce profound clinical efficacy compared to single target neutralization in a group of migraine/headache domain patients.

Both eplerizumab and LuAG09222 are genetically engineered humanized antibodies to human CGRP and PACAP, respectively, and eplerizumab and LuAG09222 can bind to these neuropeptides, thereby blocking their binding to the corresponding receptors and thus inhibiting downstream signaling that can lead to headache and pain sensation.

These antibodies are IgG1 kappa immunoglobulins comprising human constant regions. Their light and heavy chain variable regions consist of human sequences and humanized rabbit sequences.

Eplerizumab is a prescribed drug for the prophylactic treatment of migraine in adults. It is administered as 100 or 300 mg intravenous infusion (iv) therapy over 30 minutes. The patient is given 4 doses per year (i.e., one dose every 3 months).

LuAG09222 binds human PACAP with high affinity. LuAG09222 blocks migraine-associated effects (photophobia, vasodilation, elevated temperature, and lacrimation) and can alleviate pain in preclinical mechanism models. Data from clinical studies confirm the safety, tolerability and PK of LuAG 09222. Human challenge studies demonstrated that LuAG09222 prevented PACAP 38-induced vasodilation and prevented PACAP 38-induced facial flushing and heart rate increase, and that a phase 2 study of LuAG09222 in migraine (HOPE study) provided evidence of the efficacy of targeted PACAP in migraine prophylaxis. The data from the stage 2 HOPE test (NCT 05133323) underscores the role of Lu AG09222 as a potential prophylactic for migraine. In summary, the trial was consistent with its primary endpoint, with significant inter-group differences observed in the high dose group of patients receiving treatment during the 12 week double blind period. Multinational, multi-site trials are characterized by 237 individuals with episodic or chronic migraine who failed the previous 2-4 preventative agents and were randomly assigned to 750 mg (n=97) or 100 mg Lu AG09222 (n=46) or placebo (n=94) for a treatment period of 4 weeks and were followed for 12 weeks of safety. At the end of the 4 week period, the investigator observed that the high dose treated and placebo groups of individuals had a 2.0 day difference in Monthly Migraine Day (MMD) reduction (95% CI, -3.5 to-0.6; p=.0106).

Development of a composition comprising two products (eplerenone and LuAG 09222) for subcutaneous administration provides additional therapeutic benefits, and different and/or superior treatment options in headache and non-headache pain disorders. Migraine (patients with inadequate anti-CGRP response or with parasympathetic symptoms, high frequency/chronic migraine, patients in need of switching/assistance against CGRP) and cluster headache-seizures include subjects with elevated circulating CGRP and PACAP levels.

In yet another embodiment of the invention, the combination therapy or composition may be used to treat, ameliorate or prevent chronic or episodic migraine, cluster headache, endometriosis or pain.

In a particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222, wherein eplerenone has a VH region as defined in SEQ ID No.: 4 and a VL as defined in SEQ ID No.: 10, and LU AG09222 has a VH region as defined in SEQ ID No.: 15 and a VL region as defined in SEQ ID No.: 20.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims formulated to maintain the bioactivity and/or storage stability of the eplerenone and LuAG09222 antibodies therein.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the biological activity and/or storage stability of the eplerenone and LuAG09222 antibodies is maintained for at least 1 month, at least 2 months, at least 3-6 months, at least 6-9 months, at least 9-12 months, or at least one year.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any preceding claim, comprising or further comprising histidine and polysorbate 80 or poloxamer 188.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, comprising or further comprising one, two or all of NaCl, sorbitol, and arginine.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, comprising eplerenone and LuAG09222 in a total concentration of 100 mg/mL to 300 mg/mL.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, comprising eplerenone and LuAG09222 in a total concentration of 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL or 300 mg/mL.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims having a ratio of eplerenone to LuAG09222 of about 1:1, 1:2, or 2:2.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the composition comprises 100-300 mg/mL of eplerenone and 50-100 mg/mL of LuAG09222.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the composition comprises 100 mg/mL of eplerenone and 50 mg/mL of LuAG09222.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein eplerenone and LuAG0922 are the only active ingredients in the composition.

The pharmaceutical composition according to any of the preceding claims, wherein the concentration of histidine ranges between 10-50 mM, optionally 20-40 mM, the concentration of poloxamer P188 ranges between 0.0025-0.0120% w/v, and the concentration of polysorbate 80 ranges between 0.005-0.05% w/v, inclusive.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the concentration of NaCl is between 10-150 mM, optionally 30-70 mM, the concentration of sorbitol (e.g. L-sorbitol) is 50-250 mM, optionally 90-180 mM, and the concentration of arginine (e.g. L-arginine) is in the range of 50-250 mM, inclusive.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the pH is between 5.0-6.8, inclusive.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the pH is 5.0, 5.5, 5.9, 6.0, 6.5, or 6.8.

The pharmaceutical composition according to any of the preceding claims, which is suitable for intravenous administration or subcutaneous administration.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, the pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, 1:1 or 1:2, and comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, poloxamer P188 0.0025-0.0120% w/v and 30-70 mM NaCl, and having a pH of about 6, optionally a pH of 5.9.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, the pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, poloxamer P188 0.0025-0.010% w/v and 25-35 mM NaCl, and having a pH of about 6, optionally pH 5.9.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, the pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, poloxamer P188 0.005-0.010% w/v and 45-55 mM NaCl, and having a pH of about 6, optionally pH 5.9.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims, wherein the ratio of eplerenone to LuAG90222 is 1:2, and the pharmaceutical composition comprises 35-45 mM histidine buffer, 90-100 mM sorbitol, poloxamer P188 0.010-0.0120% w/v and 65-75 mM NaCl, and having a pH of about 6, optionally pH 5.9.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any preceding claim for use as a medicament.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims for use in treating or preventing headache, optionally chronic or episodic migraine or cluster headache.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims for use in the treatment or prevention of pain.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims for administration monthly (every 4 weeks) or every 2 weeks.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims or use thereof, wherein eplerenone has a heavy chain as defined in SEQ ID No.: 5 or SEQ ID No.: 6 and a light chain as defined in SEQ ID No.: 11.

In another particular embodiment, the present invention provides a pharmaceutical composition comprising eplerenone and LuAG09222 according to any one of the preceding claims or use thereof, wherein LuAG09222 has a heavy chain as defined in SEQ ID No. 16 or SEQ ID No.: 26 and a light chain as defined in SEQ ID No.: 21.

In another specific embodiment, the present invention provides a method of treating or preventing headache, optionally chronic or episodic migraine or cluster headache, comprising subcutaneously or intravenously administering a combination of eplercanizumab and LuAG09222 antibody, wherein eplercanizumab has a VH region as defined in SEQ ID No.: 4 and VL as defined in SEQ ID No.: 10, and LU AG09222 has a VH region as defined in SEQ ID No.: 15 and VL region as defined in SEQ ID No.: 20, and optionally wherein administration of the antibody combination has a cumulative or synergistic effect on the number, duration and/or intensity of inhibition, alleviation or prevention of migraine attacks compared to subcutaneous or intravenous administration of eplercanizumab alone or LuAG 09222.

In another specific embodiment, the invention provides for the treatment or prophylaxis of pain comprising subcutaneously or intravenously administering a combination of eplerenone and a LuAG09222 antibody, wherein the eplerenone has a VH region as defined in SEQ ID No.: 4 and VL as defined in SEQ ID No.: 10, and LU AG09222 has a VH region as defined in SEQ ID No.: 15 and VL region as defined in SEQ ID No.: 20, and optionally wherein the administration of the antibody combination has an additive or synergistic effect in inhibiting or alleviating pain as compared to subcutaneous or intravenous administration of eplerenone alone or LuAG 09222.

In another particular embodiment, the present invention provides a method of treating or preventing headache, optionally chronic or episodic migraine or cluster headache by administering eplerenone and LuAG09222, wherein the eplerenone and LuAG09222 antibodies are administered subcutaneously or intravenously using the pharmaceutical compositions as described above.

In another particular embodiment, the invention provides a method of treating or preventing pain (e.g., acute pain, chronic pain, neuropathic pain, nociceptive pain, and/or neuropathic pain) by administering eplerenone and LuAG09222 using a pharmaceutical composition according to the foregoing, subcutaneously or intravenously administering eplerenone and LuAG09222 antibodies.

Drawings

Figure 1 shows the number of subjects treated with Ab6 (treatment group) or placebo group in the human clinical trial described in example 1, who reduced migraine by 50%, 75% or 100% at each monitoring point throughout the time period. The right column of each group corresponds to patients receiving 1000 mg Ab6, while the left column of each group corresponds to a matched placebo control. In each response rate group, the response rate of Ab 6-receiving patients was significantly higher than placebo-treated controls, with p-values for each group as indicated at 0.0155, 0.0034 and 0.0006, respectively.

Figure 2 shows the median (±qr) change% from baseline for the monthly migraine days in the placebo and Ab6 treated groups over 12 weeks post-treatment. (p=0.0078). The upper and lower lines show the results for placebo-treated control and patients administered 1000 mg of Ab6, respectively.

Figure 3 shows the median (±qr) change% of the baseline from the number of migraine attacks per month over 12 weeks after treatment for placebo and Ab6 treated groups. The upper and lower lines show the results for placebo-treated control and patients administered 1000 mg of Ab6, respectively.

Figure 4 shows the median (±qr) change% of the number of migraine hours per month from baseline over 12 weeks after treatment for placebo and Ab6 treated groups. The upper and lower lines show the results for placebo-treated control and patients administered 1000 mg of Ab6, respectively.

Figure 5 summarizes the screening of patients, assigning to treatment and control groups and patient loss by follow-up.

Figure 6 compares HIT-6 responder analysis for Ab6 treated group and placebo group at baseline, week 4 post-treatment, week 8 post-treatment, and week 12 post-treatment.

Figure 7 shows HIT-6 analysis indicates that the effect of headache was only "some" or "few/none" patient percentages after baseline and Ab6 administration. At baseline, most patients are affected by migraine headache, either "substantial" or "severe". At each subsequent time point, the percentage of patients who were administered 1000 mg Ab6 (left column of each group, blue) were significantly higher with only "some" or "little/no" HIT-6 effect than placebo control (right column of each group, red).

Figure 8 includes Pharmacokinetic (PK) profiles of Ab6 administered intravenously at a single dose of 1000 mg.

Figure 9 includes plasma free Pharmacokinetic (PK) parameters N (patient number), mean and Standard Deviation (SD) for Ab6 at a single intravenous dose of 1000 mg. The parameters and units shown in the tables are C _max（µg/mL）、AUC_0-∞ (mgHr/mL), half-life (days), V _z (L), and C _L (mL/hr).

Figure 10 shows the monthly migraine day change (mean + -SEM) change of Ab6 (1000 mg i.v.) from baseline relative to placebo for a single dose of the study described in example 1.

FIG. 11 shows the average migraine day (+/-SD) over time for the entire analysis population of the study described in example 1. The visit interval (where the electronic diary (eDiary) was completed for 21-27 days) was normalized by multiplying the observed frequency by the reciprocal of the completion rate.

Figure 12 shows the distribution and variation of actual migraine days for Ab6 treated groups over weeks 1-4 of the study described in example 1.

Figure 13 shows the distribution and variation of the actual migraine days of the placebo group during weeks 1-4 of the study described in example 1.

Figure 14 shows the distribution and variation of actual migraine days for Ab6 treated groups over weeks 5-8 of the study described in example 1.

Figure 15 shows the distribution and variation of the actual migraine days of the placebo group in weeks 5-8 of the study described in example 1.

Figure 16 shows the distribution and variation of actual migraine days for Ab6 treated groups over weeks 9-12 of the study described in example 1.

Figure 17 shows the distribution and variation of the actual migraine days of the placebo group in weeks 9-12 of the study described in example 1.

Figure 18 shows the 50% responder rates for Ab6 and placebo treated groups studied as described in example 1. Subjects with a migraine frequency decrease of 50% or more were considered 50% responders. The visit interval (where the electronic diary was completed for 21-27 days) was normalized by multiplying the observed frequency by the inverse of the completion rate.

Figure 19 shows the 75% responder rate for Ab6 and placebo treated groups studied as described in example 1. Subjects with a migraine frequency decrease of > 75% were considered 75% responders. Normalization was performed as described in fig. 18.

Figure 20 shows 100% responder rates for Ab6 and placebo treated groups studied as described in example 1. Subjects with 100% reduction in migraine frequency were considered 100% responders. Normalization was performed as described in fig. 18.

Figure 21 shows the average migraine severity over time for the entire analysis population for the study described in example 1. On the scale used, an average score of 3 for migraine pain represents "moderate pain".

Figure 22 summarizes the change from baseline in the measured properties of placebo and treatment groups in the study described in example 1.

Figure 23 shows the percentage of migraine patients in the clinical trial described in example 2 on days 1, 7, 14, 21 and 28 in the 300 mg, 100 mg and placebo treated groups. The top row shows placebo results, the bottom row shows 300 mg doses and the middle row shows 100 mg doses.

Figure 24 shows the percentage of patients reaching a 50% reduction in migraine days in the clinical trial described in example 2 in the 300 mg and 100 mg treatment groups at month 1, month 1-3 (after 1 st infusion) and month 4-5 (after 2 nd infusion). In each figure, the data bars show the results of 100 mg, 300 mg and placebo groups from left to right. Statistical significance is shown below. ++ means statistically significant difference from placebo; + represents statistically significant differences from placebo (unregulated), and ≡represents statistically significant differences from placebo (post-analysis).

Figure 25 shows the percentage of patients in the clinical trial described in example 2 who reached a 75% reduction in migraine day in the 300 mg and 100 mg treatment groups at month 1, month 1-3 (after 1 infusion) and month 4-5 (after 2 infusions). The data sequence and statistical significance signature are shown in figure 24.

Figure 26 shows the percentage of patients reaching a 100% reduction in migraine days in the clinical trial described in example 2 in the 300 mg and 100 mg treatment groups at month 1, month 1-3 (after 1 st infusion) and month 4-5 (after 2 nd infusion). The data sequence and statistical significance signature are shown in figure 24.

Figure 27 summarizes the characteristics of the patients in each treatment group in the clinical trial described in example 3.SD, standard deviation, BMI, body Mass index according to American Academy of Neurology/American Headache Society guidelines for migraine preventative treatment [ guidelines for preventive treatment of migraine by the American Association of neurological/headache in the United states (drugs determined by clinical examination of encoded medical data).

Fig. 28, variation of Mean Migraine Day (MMD) over baseline versus placebo over 1-3 months by baseline subset of human clinical trials for chronic migraine patients. In the graph, the data points are averages and the lines show 95% Confidence Intervals (CIs) for the change from placebo for each subgroup marked on the far left, either 100 mg (upper line) or 300 mg (lower line) treatment group.

Fig. 29, variation of Mean Migraine Day (MMD) over baseline versus placebo over 1-3 months by baseline subset of human clinical trials for narcotic migraine patients.

Fig. 30, change in Mean Migraine Day (MMD) from baseline across 2 dose intervals in chronic migraine patients on acute medications on at least 1 day per month at baseline. Triangle, placebo (n=366). Round 100 mg Ab6 (n=356) per dose. Square 300 mg Ab6 (n=350) per dose.

Fig. 31, average days of acute drug use in chronic migraine patients who used acute drugs at least one day per month at baseline. Triangle, placebo (n=366). Round 100 mg Ab6 (n=356) per dose. Square 300 mg Ab6 (n=350) per dose.

Fig. 32 changes from baseline in acute drug use for a subgroup of chronic migraine patients with different days of acute drug use at baseline. Solid line: patients who used acute drugs 10 days or more per month at baseline. Dashed line-patients on acute medications at least 1 day and less than 10 days per month at baseline. Triangle, placebo. Round 100 mg Ab6 per dose. Square 300 mg Ab6 per dose.

Fig. 33 summary of acute drug days for a subgroup of chronic migraine patients with baseline acute drug use.

Fig. 34, average migraine day (MMD) changes from baseline across 2 dose intervals in episodic migraine patients on acute medications on at least 1 day per month at baseline. Triangle, placebo (n=222). Circles 100 mg Ab6 (n=221) per dose. Square 300 mg Ab6 (n=222) per dose.

Fig. 35 average days of acute drug use in episodic migraine patients who used acute drug at least one day per month at baseline. Triangle, placebo (n=222). Circles 100 mg Ab6 (n=221) per dose. Square 300 mg Ab6 (n=222) per dose.

Fig. 36 changes from baseline in acute drug use for a subset of narcotic migraine patients with different days of acute drug use. Solid line: patients who used acute drugs 10 days or more per month at baseline. Dashed line-patients on acute medications at least 1 day and less than 10 days per month at baseline. Triangle, placebo. Round 100 mg Ab6 per dose. Square 300 mg Ab6 per dose.

Fig. 37 summary of acute drug days for a subset of patients with narcotic migraine who had baseline acute drug use.

Fig. 38 day-1 was included in migraine data. Day 0 was defined as the infusion day. Thus, day 0 data indicates the therapeutic effect following infusion.

Fig. 39 based on clinical data from LuAG09222 (Ab 10.h3) trial, particularly phase II trial from migraine sufferers, dose prediction has been performed using R software (nlmixr). Monthly dosing, for 7 months, then discontinued.

Detailed Description

Combining the population PK model of eplerenone and LuAG09222 allows the establishment of a PKPD model to determine the ratio of eplerenone and LuAG09222 required to have less than 50% of each target (CGRP and PACAP) free. For both compounds, the administration was explored as simultaneous bolus administration. The dosing frequency is defined as once a month and the route of administration is subcutaneous.

Eplerizumab has a terminal elimination half-life of 27 days and is used at 100 or 300 mg in migraine, while LuAG09222 has a terminal elimination half-life of about 15 days.

If less than 50% of free target is to be obtained at monthly s.c. dosing intervals, it is estimated that 100 mg doses of eplerenone and 50 mg doses of LuAG09222 are required.

When administered as a single agent, no treatment-related death or adverse outcome due to pharmacological activity of eplerian mab or LuAG09222 was observed during single-dose or repeated-dose studies in rats or cynomolgus monkeys. No effect on reproductive function or performance, fertility or early embryo development of rats was observed following eplerizumab administration. For eplerenone or LuAG09222 administration, no evidence of parental effects or embryonic lethality, fetal toxicity, or teratogenicity was observed in the rats or rabbits. During prenatal and postnatal development studies with eplerizumab in rats, there was no effect on survival, physical development, behaviour or reproductive performance of the F1 generation.

In all non-clinical studies, NOEL/NOAEL with either eplerizumab or LuAG09222 administered as a single agent was the highest dose administered (up to 150 mg/kg/dose).

Since eplerenone and LuAG09222 are both humanized monoclonal antibodies, administration or co-administration of the combination product is not expected to produce pharmacokinetic interactions.

Definition of the definition

It is to be understood that this invention is not limited to the particular methods, protocols, cell lines, animal species or genera, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "chronic migraine" refers to a condition in which a patient exhibits an average of at least 15 headaches per month, wherein a subset of these headache days meet the ICHD-3 criteria for migraine with or without aura. The term "episodic migraine" refers to a condition in which the patient exhibits an average monthly headache of less than 15 days, wherein typically 4-15 is a migraine phenotype conforming to the definition of ICHD-3 for migraine with or without aura.

As used herein, the term "diagnosing chronic migraine" refers to a patient who meets clinical criteria for chronic migraine, whether or not the patient is formally diagnosed. The term "diagnosing narcotic migraine" refers to a patient meeting clinical criteria for narcotic migraine, whether or not the patient is formally diagnosed.

As used herein, the term "intravenous administration" refers to a mode of administration in which a substance, such as an antibody, is introduced directly into the circulation of a patient, most typically into the venous circulation. The substance may be introduced into a carrier liquid, optionally an aqueous solution, such as physiological saline. The substance may be administered as a single formulation or as multiple formulations, so long as administration is completed within a short period of time (e.g., within 1 day, preferably within 12 hours, more preferably within 6 hours, and most preferably within 1-2 hours). The term "subcutaneous administration" (or s.c. administration) refers to a mode of administration in which a substance (e.g., an antibody) is applied to a layer of skin called epidermis, just below the dermis layer and epidermis layer. Subcutaneous administration can be applied to a variety of sites including the outer region of the upper arm and the upper hip region behind the abdomen, thigh front, upper back, or hip bone.

As used herein, the term "baseline migraine days" refers to the number of migraine days a patient exhibits during a specified period of time, for example, prior to treatment. For example, baseline migraine days may be determined over a period of one month or more, such as by recording whether migraine occurred daily.

As used herein, the term "monthly migraine day" refers to the number of days a patient experiences migraine monthly, i.e., at any time of the day, the patient has symptoms that are in accordance with the clinical definition of migraine. The number of migraine days per month can be determined by recording whether migraine occurred daily.

As used herein, the term "monthly headache day" refers to the number of days that a patient experiences headache monthly, i.e., at any time during the day, the patient has symptoms that are in accordance with the clinical definition of headache. The number of headache days per month can be determined by recording whether headache occurred per day.

Calcitonin Gene Related Peptide (CGRP) As used herein, CGRP encompasses the following homo sapiens CGRP-alpha and homo sapiens CGRP-beta amino acid sequences obtainable from American peptide company (AMERICAN PEPTIDES) (Sunnyvale Calif.) and Bachem company (Bachem) (Torons, calif.).

CGRP-alpha ACDTATCVTHRLAGLLSRSGGVVKNNFVP TNVGSKAF-NH ₂ (SEQ ID NO: 22) in which the terminal phenylalanine is amidated;

CGRP-beta ACNTATCVTHRLAGLLSRSGGMVKSNFV PTNVGSKAF-NH ₂ (SEQ ID NO: 23) in which the terminal phenylalanine is amidated, and also covers any membrane-bound form of these CGRP amino acid sequences, as well as mutants (mutants), splice variants, isoforms, orthologs, homologs and variants of the sequences.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) as used herein, unless otherwise indicated, PACAP includes any mammalian form of PACAP, and specifically encompasses the following homo sapiens PACAP27 and homo sapiens PACAP38 amino acid sequences.

PACAP38:

HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQR VKNK (SEQ ID NO: 24), and also includes any mutants, splice variants, isoforms, orthologs, homologs and variants of the sequence.

PACAP27:

HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 25), and also includes any mutants, splice variants, isoforms, orthologs, homologs and variants of the sequence.

Expression vectors these DNA vectors contain elements that facilitate the expression of the foreign protein in a target host cell, such as a yeast or mammalian cell, optionally a Pichia pastoris or CHO cell. Conveniently, manipulation of the sequences and production of DNA for transformation is first carried out in a bacterial host, such as e.coli, and typically the vector will include sequences that facilitate such manipulation, including a bacterial origin of replication and a suitable bacterial selectable marker. The selectable marker encodes a protein necessary for survival or growth of the transformed host cell grown in the selective medium. Host cells not transformed with a vector containing the selection gene will not survive in the medium. Typical selection genes encode proteins that (a) are resistant to antibiotics or other toxins, (b) are auxotrophic, or (c) provide key nutrients that are not available from complex media. Exemplary vectors and methods for transforming yeast are described in, for example, ,Burke, D., Dawson, D., & Stearns, T. (2000). Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual [ methods of Yeast genetics, cold spring harbor laboratory curriculum, prain's View, N.Y.: cold Spring Harbor Laboratory Press, cold spring harbor laboratory Press.

Expression vectors for yeast or mammalian cells will typically further include yeast or mammalian specific sequences, including selective auxotrophs or drug markers for identifying transformed yeast strains or transformed mammalian cells. Drug markers can further be used to amplify the copy number of the vector in the host cell.

The polypeptide coding sequence of interest is operably linked to transcriptional and translational regulatory sequences that provide for the expression of the polypeptide in a host cell, such as Pichia pastoris or CHO cells. These vector components may include, but are not limited to, one or more of an enhancer element, a promoter, and a transcription termination sequence. Sequences for secretion of polypeptides, such as signal sequences and the like, may also be included. The origin of replication of the yeast or mammal is optional, as the expression vector is typically integrated into the host cell genome. In one embodiment of the invention, the polypeptide of interest is operably linked or fused to a sequence that provides for optimal secretion of the polypeptide from a yeast diploid cell.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a signal sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the signal sequence is operably linked to the DNA of the polypeptide, and if the promoter or enhancer affects the transcription of the sequence, it is operably linked to the coding sequence. In general, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Enhancers need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites or alternatively by PCR/recombination methods familiar to those skilled in the art (Gateway ^R technology; invitrogen, calif.). If such a site is not present, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

A promoter is an untranslated sequence (typically within about 100 bp to 1000 bp) located upstream (5') of the structural gene start codon that controls the transcription and translation of a particular nucleic acid sequence to which it is operably linked. Such promoters fall into several categories, inducible, constitutive and repressible (increased transcription levels in response to lack of repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to certain changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature).

Promoter fragments may also be used as sites for homologous recombination of the expression vector and integration into the host genome at the same site, alternatively selectable markers may be used as sites for homologous recombination. Examples of suitable promoters from Pichia include AOX1 and promoters (Cregg et al (1989) mol. Cell. Biol. [ molecular and cell biology ] 9:1316-1323), ICL1 promoter (Menendez et al (2003) Yeast [ Yeast ] 20 (13): 1097-108), glyceraldehyde 3-phosphate dehydrogenase promoter (GAP) (Waterham et al (1997) Gene [ Gene ] 186 (1): 37-44), and FLD1 promoter (Shen et al (1998) Gene [ Gene ] 216 (1): 93102). GAP promoter is a strong constitutive promoter, while AOX and FLD1 promoters are inducible.

Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, pyk, and chimeric promoters derived therefrom. In addition, non-yeast promoters may be used in the present invention, optionally mammalian, insect, plant, reptile, amphibian, viral and avian promoters. Most typically, the promoter will comprise a mammalian promoter (which may be endogenous to the expressed gene) or will comprise a yeast or viral promoter that provides efficient transcription in a yeast system.

Examples of mammalian promoters include Cytomegalovirus (CMV) -derived promoters, chicken 3-actin (CBM) -derived promoters, adenomatous Polyposis Coli (APC) -derived promoters, leucine-rich repeat-rich G protein-coupled receptor 5 (LGR 5) promoters, CAG promoters, beta actin promoters, elongation factor 1 (EF 1) promoters, early growth response 1 (EGR-1) promoters, eukaryotic initiation factor 4A (EIF 4A 1) promoters, monkey virus 40 (SV 40) early promoters, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoters, moMuLV promoters, epstein-barr virus immediate early promoters, rous sarcoma virus promoters, and human gene promoters (optionally but not limited to actin promoters, myosin promoters, hemoglobin promoters and creatine kinase promoters), among others. Combinations of two or more of the foregoing promoters may also be used. In addition, inducible promoters may be used. The use of inducible promoters provides a molecular switch capable of initiating expression of a polynucleotide sequence that is operably linked when such expression is desired or that shuts down expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The antibodies of the invention can be produced recombinantly not only directly, but also as fusion polypeptides with heterologous polypeptides (e.g., signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide). In general, the signal sequence may be a component of a vector, or may be part of a polypeptide coding sequence inserted into a vector. Preferably, the heterologous signal sequence selected is a sequence that is recognized and processed by one of the standard pathways available in the host cell. The Saccharomyces cerevisiae alpha factor prepro signal has been shown to be effective in secreting various recombinant proteins from Pichia pastoris. Other yeast signal sequences include alpha mating factor signal sequences, invertase signal sequences, and signal sequences derived from other secreted yeast polypeptides. In addition, these signal peptide sequences can be engineered to provide enhanced secretion in diploid yeast expression systems. Secretion signals for mammalian as well as yeast cells include mammalian signal sequences, which may be heterologous to the secreted protein or may be native to the secreted protein. The signal sequence includes a propeptide (pre-peptide) sequence and, in some cases, a propeptide (propeptide) sequence. Many such signal sequences are known in the art, including those found on immunoglobulin chains, such as K28 protoxin sequences, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chains, human Ig light chains, and the like. See, for example, hashimoto et al Protein Eng [ Protein engineering ] 11 (2) 75 (1998), and Kobayashi et al Therapeutic Apheresis [ therapeutic isolation ] 2 (4) 257 (1998).

Transcription can be increased by inserting a transcription activation sequence into the vector. These activators are cis-acting elements of DNA, typically about 10 bp to 300 bp, which act on the promoter to increase its transcription. Transcriptional enhancers are relatively independent in orientation and position within introns as well as within the coding sequence itself, found 5 'and 3' of the transcriptional unit. Enhancers may be spliced into the expression vector at the 5' or 3' position of the coding sequence, but are preferably located at the 5' site of the promoter.

Expression vectors used in eukaryotic host cells may also contain sequences necessary to terminate transcription and stabilize mRNA. In the untranslated region of eukaryotic or viral DNA or cDNA, such sequences are typically available from 3' to the translation stop codon. These regions comprise nucleotide fragments transcribed into polyadenylation fragments in the untranslated portion of the mRNA.

Construction of a suitable vector containing one or more of the above components uses standard ligation techniques or PCR/recombinant methods. The isolated plasmid or DNA fragment is cut, trimmed and religated either to the desired form used to produce the desired plasmid or by recombinant means. To perform an analysis to confirm the correct sequence in the constructed plasmid, the ligation mixture is used to transform host cells and successful transformants are selected by antibiotic (e.g., ampicillin or bleomycin) resistance where appropriate. Plasmids from transformants were prepared and analyzed and/or sequenced by restriction endonuclease digestion.

As an alternative to restriction and ligation of fragments, recombination methods based on att sites and recombinases can be used to insert DNA sequences into vectors. Such methods are described, for example, by Landy (1989) Ann. Rev. Biochem. [ annual biochemistry ] 58:913-949, and are known to those of skill in the art. Such a method utilizes intermolecular DNA recombination mediated by a mixture of lambda and a recombinant protein encoded by Escherichia coli. Recombination occurs between specific attachment (att) sites on interacting DNA molecules. For a description of att sites, see Weisberg and Landy (1983) Site-Specific Recombination in Phage Lambda, in Lambda II [ Site-specific recombination of phage Lambda in Lambda II ], edit Weisberg (Cold spring harbor (Cold Spring Harbor), new York: cold Spring Harbor Press [ Cold spring harbor Press ]), pages 211-250. The DNA fragments flanking the recombination sites are switched such that the recombined att sites are heterozygous sequences consisting of the sequences supplied by each parental vector. Recombination can occur between DNA of any topology.

The att site may be introduced into a sequence of interest by ligating the sequence of interest into a suitable vector, generating a PCR product containing the att B site by using specific primers, generating a cDNA library cloned into a suitable vector containing the att site, etc.

Folding, as used herein, refers to the three-dimensional structure of polypeptides and proteins in which interactions between amino acid residues play a role in stabilizing the structure. Appropriate folding is typically the arrangement of polypeptides that results in optimal biological activity, and in the case of antibodies, can be conveniently monitored by assays of activity (e.g., antigen binding).

The expression host may be further modified by introducing sequences encoding one or more enzymes that enhance folding and disulfide bond formation, i.e., folding enzymes, chaperones, and the like. Such sequences may be expressed constitutively or inducibly in the yeast host cell using vectors, markers, etc. as known in the art. Preferably, the sequence comprising transcriptional regulatory elements sufficient to achieve the desired expression pattern is stably integrated into the yeast genome by a targeting method.

For example, eukaryotic PDIs are not only effective catalysts for protein cysteine oxidation and disulfide isomerization, but also exhibit chaperone activity. Co-expression of PDI can facilitate the production of active proteins with multiple disulfide bonds. Expression of BIP (immunoglobulin heavy chain binding protein), cyclophilin, and the like is also of interest. In one embodiment of the invention, each haploid parent strain expresses a different folding enzyme, e.g., one strain may express BIP and another strain may express PDI or a combination thereof.

The terms "desired protein" or "desired antibody" are used interchangeably and generally refer to a parent antibody specific for a target, i.e., CGRP, PACAP, or chimeric or humanized antibodies or derived binding portions thereof as described herein. The term "antibody" is intended to include any polypeptide chain-containing molecular structure having a specific shape that fits and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Prototype antibody molecules are immunoglobulins, in particular IgG from all sources, e.g. human, rodent, rabbit, bovine, ovine, porcine, canine, other mammalian, chicken, other avian, etc., which are considered "antibodies". Many antibody coding sequences have been described and others may be proposed by methods well known in the art. Examples include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (e.g., scFv), camelbody, nanobody, igNAR (single chain antibody derived from shark), small Modular Immunopharmaceuticals (SMIPs), and antibody fragments (e.g., fab ', F (ab') ₂, etc.). See Streltsov VA et al , Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype [ modeling of the structure and early developmental isotype of shark IgNAR antibody variable domain [ Protein sci ] [ Protein science ] month 11 2005; 14 (11): electronic publication of 30 th month of 2901-9.2005, greenberg AS et al , A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks [ new antigen receptor gene family which underwent rearrangement and extensive somatic diversification in sharks, [ Nature ] 3 rd month of 1995, 374 (6518): 168-73; nuttall SD et al , Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries [ isolated new antigen receptors from Wobbe Gong Sha and used AS scaffolds for displaying Protein loop libraries, [ molecular immunology ] 2001, 38 (4): 313-26; hamers-CASTERMAN C et al, naturally occurring antibodies devoid of LIGHT CHAINS [ natural antibodies free of light chains ], nature [ Nature ] 6 th month of 1993 ], 363 (6428): 446-8; gill DS et al Biopharmaceutical drug discovery using novel Protein scaffolds [ biopharmaceutical discovery using novel Protein scaffolds ], curr Opin Biotechnology current view ] 12 months, 17 (6): electronic publication of 19 th month of 2006-8.2006.

For example, antibodies or antigen binding fragments may be produced by genetic engineering. In this technique, as with other methods, the antibody-producing cells are sensitive to the desired antigen or immunogen. Messenger RNA isolated from antibody-producing cells was used as a template to prepare cDNA by PCR amplification. Libraries of vectors are generated by inserting appropriate portions of the amplified immunoglobulin cDNA into expression vectors, each vector comprising a heavy chain gene and a light chain gene that retains the original antigen specificity. A combinatorial library is constructed by combining a heavy chain gene library with a light chain gene library. This resulted in a clone library (similar to Fab fragments or antigen binding fragments of antibody molecules) that co-expressed heavy and light chains. Vectors carrying these genes are co-transfected into host cells. When antibody gene synthesis is induced in transfected hosts, the heavy and light chain proteins self-assemble to produce active antibodies, which can be detected by screening with antigen or immunogen.

Antibody coding sequences of interest include those encoded by natural sequences, as well as those encoded by nucleic acids and variants thereof that differ from the disclosed nucleic acid sequences due to the degeneracy of the genetic code. Variant polypeptides may include amino acid (aa) substitutions, additions or deletions. Amino acid substitutions may be conservative amino acid substitutions or substitutions that eliminate non-essential amino acids, such as altering the glycosylation site, or minimizing misfolding by substituting or deleting functionally unnecessary one or more cysteine residues. Variants can be designed to retain or enhance the biological activity of a particular region of a protein (e.g., a functional domain, catalytic amino acid residue, etc.). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. In vitro mutagenesis techniques for cloned genes are known. The invention also includes polypeptides modified using common molecular biology techniques to increase their resistance to proteolytic degradation or to optimize solubility or make them more suitable as therapeutic agents.

Chimeric antibodies may be prepared by recombinant means that combine variable light and heavy chain regions (V _L and V _H) obtained from antibody-producing cells of one species, while constant light and heavy chain regions are from another species. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions to produce antibodies having predominantly human domains. The production of such chimeric antibodies is well known in the art and may be accomplished by standard means (e.g., as described in U.S. Pat. No. 5,624,659, which is incorporated herein by reference in its entirety). It is further contemplated that the human constant region of the chimeric antibodies of the invention may be selected from the group consisting of IgG1, igG2, igG3, and IgG4 constant regions.

Humanized antibodies are engineered to contain even more human-like immunoglobulin domains and incorporate only the complementarity determining regions of animal-derived antibodies. This is accomplished by carefully examining the sequences of the hypervariable loops of the variable regions of the monoclonal antibodies and adapting them to the structure of the human antibody chain. Although seemingly complex, the process is well defined in practice. See, for example, U.S. Pat. No. 6,187,287, which is fully incorporated by reference herein.

In addition to intact immunoglobulins (or recombinant counterparts thereof), immunoglobulin fragments (e.g., fab ', F (ab') ₂ or other fragments) comprising an epitope binding site may also be synthesized. "fragments" or minimal immunoglobulins can be designed using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the present invention may be produced by synthesizing fused variable light and variable heavy chain regions. Combinations of antibodies are also of interest, e.g., diabodies, which comprise two different Fv specificities. In another embodiment of the invention, immunoglobulin fragments encompass SMIPs (small molecule immune drugs), camelid antibodies, nanobodies and ignars.

Immunoglobulins and fragments thereof may be post-translationally modified, e.g., to add effector moieties, e.g., chemical linkers, detectable moieties, e.g., fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials, chemiluminescent moieties, etc., or specific binding moieties, e.g., streptavidin, avidin, biotin, etc., which may be used in the methods and compositions of the invention. Examples of additional effector molecules are provided below.

A polynucleotide sequence "corresponds" to a polypeptide sequence if translation of the polynucleotide sequence according to the genetic code produces a polypeptide sequence (i.e., the polynucleotide sequence "encodes" a polypeptide sequence), and one polynucleotide sequence "corresponds" to another polynucleotide sequence if the two polynucleotide sequences encode the same polypeptide sequence.

The "heterologous" region or domain of a DNA construct is a recognizable fragment of DNA in a large DNA molecule that is not found in nature in association with the large molecule. Thus, when a heterologous region encodes a mammalian gene, the gene is typically flanked by DNA that does not flank mammalian genomic DNA in the genome of the source organism. Another example of a heterologous region is a construct in which the coding sequence itself (e.g., a cDNA in which the genomic coding sequence comprises an intron, or a synthetic sequence having codons that differ from the native gene) is not found in nature. Allelic variation or naturally occurring mutation events do not result in heterologous regions of DNA as defined herein.

A "coding sequence" is an in-frame sequence of codons that corresponds to or encodes (in terms of the genetic code) a protein or peptide sequence. If the sequences or their complements encode the same amino acid sequence, the two coding sequences correspond to each other. Coding sequences associated with appropriate regulatory sequences may be transcribed and translated into polypeptides. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence. A "promoter sequence" is a DNA regulatory region that is capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for binding regulatory molecules (e.g., transcription factors) that affect transcription of the coding sequence. When an RNA polymerase binds to a promoter sequence in a cell and transcribes the coding sequence into mRNA (which is then translated into a protein encoded by the coding sequence), the coding sequence is "under the control" or "operably linked" to the promoter sequence.

Vectors are used to introduce foreign substances (e.g., DNA, RNA, or proteins) into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for polypeptides). A "DNA vector" is a replicon, such as a plasmid, phage, or cosmid, to which another polynucleotide segment may be attached so that the attached segment replicates. An "expression vector" is a DNA vector that comprises regulatory sequences that will direct the synthesis of a polypeptide by an appropriate host cell. This generally means a promoter that binds to RNA polymerase and initiates transcription of the mRNA, as well as a ribosome binding site and initiation signal to direct the conversion of the mRNA into one or more polypeptides. The polynucleotide sequence is incorporated into an expression vector at the correct site and in the correct reading frame, and the vector is then transformed into a suitable host cell such that the polypeptide encoded by the polynucleotide sequence is produced.

"Amplification" of a polynucleotide sequence is the in vitro production of multiple copies of a particular nucleic acid sequence. The amplified sequence is typically in the form of DNA. A number of techniques for performing such amplification are described in the review article by Van Brunt (1990, bio/technology, [ Bio/technology ], 8 (4): 291-294). Polymerase chain reaction or PCR is a prototype of nucleic acid amplification, and the use of PCR herein should be considered as an example of other suitable amplification techniques.

The general structure of antibodies in vertebrates is now well understood (Edelman, g.m., ann.n.y. Acad. Sci. [ new york academy of sciences, annual book ], 190:5 (1971)). Antibodies consist of two identical light chain polypeptides ("light chains") having a molecular weight of about 23,000 daltons and two identical heavy chains ("heavy chains") having a molecular weight of 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain loops around the heavy chain starting at the opening in the "Y" configuration. The "branched" portion of the "Y" configuration is referred to as the F _ab region, and the stem portion of the "Y" configuration is referred to as the F _C region. The amino acid sequence is oriented from the N-terminus at the top of the "Y" configuration to the C-terminus at the bottom of each strand. The N-terminus has a variable region specific for the antigen that elicits it, and the variable region is approximately 100 amino acids in length, with minor variations in length between the light and heavy chains and between different antibodies.

The variable region is linked in each chain to a constant region that extends the remaining length of the chain, and the constant region in a particular class of antibodies does not vary with the specificity of the antibody (i.e., the antigen that elicits it). There are five main classes of known constant regions that determine the class of immunoglobulin molecules (IgG, igM, igA, igD and IgE correspond to gamma, mu, alpha, delta and epsilon heavy chain constant regions). The constant region or class determines the subsequent effector functions of the antibody, including complement activation (Kabat, E.A., structural Concepts in Immunology and Immunochemistry [ structural concepts in Immunology and immunochemistry ], 2 nd edition, pages 413-436, holt, rinehart, winston [ Hall, leishat and Wenston Press ] (1976)), and other cellular responses (Andrews, D.W., et al, clinical Immunobiology [ clinical immunobiology ], pages 1-18, W.B., sanders [ W.B., morus publishing company ] (1980); kohl, S. et al, immunology [ Immunology ], 48:187 (1983)), while the variable region determines the antigen to be reacted with. Light chains are classified as either kappa or lambda. Each heavy chain may be prepared with either a kappa or lambda light chain. When immunoglobulins are produced by hybridomas or by B cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds.

The expression "variable region" or "VR" refers to the domain within each pair of light and heavy chains in an antibody that is directly involved in the binding of the antibody to an antigen. Each heavy chain has a variable domain (V _H) at one end followed by multiple constant domains. Each light chain has a variable domain (V _L) at one end and a constant domain at the other end, the constant domain of the light chain being aligned with the first constant domain of the heavy chain and the light chain variable domain being aligned with the heavy chain variable domain.

The expression "complementarity determining region", "hypervariable region" or "CDR" refers to one or more hypervariable or Complementarity Determining Regions (CDRs) found in the variable region of the light or heavy chain of an antibody (see Kabat, e.a. et al Sequences of Proteins of Immunological Interest [ protein sequences of immunological significance ], national Institutes of Health [ national institutes of health ], bezidas (Bethesda), maryland, (1987)). These include hypervariable regions defined by Kabat et al ("Sequences of Proteins of Immunological Interest [ protein sequences having immunological significance ]," Kabat E. Et al, U.S. Dept. Of HEALTH AND Human Services [ U.S. department of health and public service ], 1983 ] or hypervariable loops of antibody 3-dimensional structure (Chothia and Lesk, J mol. Biol. [ J. Mol. Biol. Mol. J. 196 901-917 (1987)). The CDRs in each chain are tightly linked by a framework region and together with the CDRs in the other chain contribute to the formation of antigen binding sites. In the CDRs, there are selected amino acids described as Selective Determining Regions (SDRs) which represent the critical contact residues used by CDRs in antibody-antigen interactions (Kashmiri, s., methods [ Methods ], 36:25-34 (2005)). In the present invention, when a particular antibody amino acid or nucleic acid residue is referred to by a number, it is generally referred to as its position in a particular amino acid or nucleic acid sequence (i.e., a particular sequence identifier) and/or numbering according to Kabat et al.

The expression "framework region" or "FR" refers to one or more framework regions within the antibody light and heavy chain variable regions (see Kabat, e.a. et al, sequences of Proteins of Immunological Interest [ protein sequences with immunological significance ], national Institutes of Health [ national institutes of health ], bezistada (Bethesda), maryland, (1987)). These expressions include those amino acid sequence regions inserted between CDRs within the antibody light and heavy chain variable regions.

"Cmax" refers to the maximum (or peak) concentration of an antibody or other compound in a test area (e.g., serum or another compartment, such as cerebrospinal fluid) that is reached after administration of a drug. For example, the serum Cmax can be measured from serum (prepared by collecting a blood sample, coagulating it by centrifugation or other means and separating the solid components to produce serum (blood free of blood cells and clotting factors)), and then detecting the concentration of the analyte in the serum by ELISA or other means known in the art.

"AUC" refers to the area under the concentration-time curve, in mg/mL, unless otherwise indicatedHr (or equivalently mg)Hr/ml) is expressed in units. "AUC _0-t" refers to the area under the concentration-time curve from time=0 to the last quantifiable concentration. "AUC _0-inf" refers to the area under the concentration-time curve extrapolated from time=0 to infinity.

"I _max" refers to the maximum pharmacodynamic response elicited by an anti-CGRP antibody dose (preferably 350 mg or more, more typically at least 750mg or 1000 mg) as compared to the response elicited by a lower anti-CGRP antibody dose, for example wherein such response can be detected by inhibiting vasodilation following topical administration of capsaicin.

Antibody Ab6 (eplerizumab) sequences

Antibody Ab6 (eplerizumab) contains a variable light chain sequence as set forth below ：QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQK PGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR（SEQ ID NO.: 10）.

Antibody Ab6 (eplerizumab) contains the light chain sequences as listed below ：QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID NO.:11）.

Antibody Ab6 (eplerizumab) contains a variable heavy chain as set forth below ：EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS（SEQ ID NO.: 4）.

Antibody Ab6 (eplerizumab) contains the heavy chain sequences as listed below ：EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO.: 6）

Alternatively, the heavy chain of Ab6 (eplerizumab) may lack a C-terminal lysine, a heavy chain sequence comprising the sequences listed below ：EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（SEQ ID NO.: 5）.

In one embodiment of the invention described herein (below), the Fab fragment may be produced by enzymatic digestion (e.g., papain) of Ab 6. In another embodiment of the invention, ab6 or Fab fragments thereof can be produced by expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungi, insects or microbial systems such as yeast cells (e.g., diploid yeast, e.g., pichia diploid) and other yeast strains. Suitable pichia species include, but are not limited to, pichia pastoris.

The CDR regions of Ab6 (eplerenone) are summarized below

Light chain CDR 1 QASQSVYHNTYLA (SEQ ID NO.: 7)

Light chain CDR 2 DASTLAS (SEQ ID NO.: 8)

Light chain CDR 3 LGSYDCTNGDCFV (SEQ ID NO.: 9)

Heavy chain CDR 1 GYYMN (SEQ ID NO.: 1)

Heavy chain CDR2 VIGINGATYYASWAKG (SEQ ID NO.: 2)

Heavy chain CDR 3 GDI (SEQ ID NO.: 3)

Antibody LuAG09222

LuAG09222 contains the variable heavy chain sequences as set forth below:

EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMT WVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSS（SEQ ID NO.: 15）

LuAG09222 contains the heavy chain sequences as listed below:

EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMT WVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（SEQ ID NO.: 16）.

according to some aspects of the invention, luAG009222 may have a terminal lysine in the heavy chain as set forth below

EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMT WVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO.: 26）

LuAG09222 contains a variable light chain sequence comprising the sequences listed below:

DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAW FQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKR（SEQ ID NO.: 20）.

LuAG09222 contains a light chain sequence comprising the sequences listed below:

DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAW FQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID NO.: 21）.

In one embodiment of the invention described herein (below), fab fragments can be produced by enzymatic digestion (e.g., papain) of ab10.h3. In another embodiment of the invention, luAG09222 can be produced by expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeast, e.g., pichia diploid) and other yeast strains. Suitable pichia species include, but are not limited to, pichia pastoris.

The CDR regions of LuAG09222 are summarized below

Light chain CDR 1 QSSESVYGNYLA (SEQ ID NO.: 17)

Light chain CDR 2 EASKLES (SEQ ID NO.: 18)

Light chain CDR 3 AGGDISEGVA (SEQ ID NO.: 19)

Heavy chain CDR 1 SYYMT (SEQ ID NO.: 12)

Heavy chain CDR 2 FIDAGGDAYYASWAKG (SEQ ID NO.: 13)

Heavy chain CDR 3 DLDL (SEQ ID NO.: 14)

Antibodies and fragments thereof may be post-translationally modified to add effector moieties, such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties, such as, for example, streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials, as described herein.

Antibodies or fragments thereof may also be chemically modified to provide additional advantages such as increased polypeptide solubility, stability and circulation time (in vivo half-life) or reduced immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moiety used for derivatization may be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. Antibodies and fragments thereof may be modified at random positions within the molecule or at predetermined positions within the molecule, and may include one, two, three, or more attached chemical moieties.

The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycols, the preferred molecular weight is from about 1 kDa to about 100 kDa (the term "about" indicates that in the preparation of polyethylene glycols, some molecules will weigh more than the molecular weight and some will weigh less) for ease of handling and manufacture. Other sizes may be used depending on the desired therapeutic properties (e.g., desired duration of sustained release, impact on biological activity (if any), ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol on a therapeutic protein or the like). For example, the polyethylene glycol may have an average molecular weight of about 200、500、1000、1500、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10,000、10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500、20,000、25,000、30,000、35,000、40,000、50,000、55,000、60,000、65,000、70,000、75,000、80,000、85,000、90,000、95,000、 or 100,000 kDa. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; morpago et al, appl. Biochem. Biotechnol. [ applied biochemistry and biotechnology ], 56:59-72 (1996), vorobjev et al, nucleosides and Nucleotides [ nucleosides and nucleotides ], 18:2745-2750 (1999), and Caliceti et al, bioconjug. Chem. [ bioconjugation chemistry ], 10:638-646 (1999), the disclosure of each of which is incorporated herein by reference.

There are many attachment methods available to those skilled in the art, see for example EP 0 401 384, which is incorporated herein by reference (coupling PEG to G-CSF), see also Malik et al, exp. Hematol [ Experimental hematology ] 20:1028-1035 (1992) (polyethylene glycol of GM-CSF using trityl chloride is reported). For example, polyethylene glycol may be covalently bound through amino acid residues via reactive groups such as free amino groups or carboxyl groups. Reactive groups are those to which activated polyethylene glycol molecules can bind. Amino acid residues having a free amino group may include lysine residues and N-terminal amino acid residues, and those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Thiol groups may also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes are attachments on amino groups, for example on the N-terminal or lysine groups.

As described above, polyethylene glycol may be attached to proteins by linkage to any of a number of amino acid residues. For example, polyethylene glycol may be linked to the polypeptide by covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. Polyethylene glycol may be attached to a particular amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof) using one or more reaction chemistry methods.

Alternatively, the antibody or fragment thereof may have an increased in vivo half-life by fusion with albumin (including, but not limited to, recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969 issued 3/2 in 1999, european patent No. 0 413 622 and U.S. Pat. No. 5,766,883 issued 6/16 in 1998, which are incorporated herein by reference in their entireties)) or other circulating blood proteins such as transferrin or ferritin. In a preferred embodiment, the polypeptides and/or antibodies of the invention (including fragments or variants thereof) are fused to a mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin, as shown in figures 1 and 2 of european patent 0 322 094), which is incorporated herein by reference in its entirety. The invention also encompasses polynucleotides encoding the fusion proteins of the invention.

With respect to the detectable moiety, additional exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Other exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein isothiocyanate, umbelliferone, dichlorotriazinamine, phycoerythrin, and dansyl chloride. Other exemplary chemiluminescent moieties include, but are not limited to, luminol. Other exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Other exemplary radioactive materials include, but are not limited to, iodine 125 (¹²⁵ I), carbon 14 (¹⁴ C), sulfur 35 (³⁵ S), tritium (³ H), and phosphorus 32 (³² P).

With respect to the functional moiety, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, alkylating agents such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carbamazepine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (p-platinum (paraplatin)), anthracyclines including daunorubicin (formerly daunomycin), doxorubicin (Adriamycin), dithimycin, erythromycin, idarubicin, epirubicin, mitoxantrone and bichrombin, antibiotics including actinomycin (C), bleomycin, calicheamicin, and mitomycin (AMC) and vincristine, such as vinblastine, and the like. Other cytotoxic agents include paclitaxel (tacazol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetidine, etoposide, teniposide, colchicine, dihydroxyanthrax-dinedione, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, methylbenzyl hydrazine, hydroxyurea, asparaginase, corticosteroids, mitotane (mytotane) (O, P' - (DDD)), interferons, and mixtures of these cytotoxic agents.

Other cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, and bleomycin. Toxic enzymes from plants and bacteria (e.g., ricin, diphtheria toxin, and pseudomonas toxin) can be conjugated to humanized antibodies or chimeric antibodies or binding fragments thereof to produce cell type specific killers (Youle et al, proc. Nat ' l Acad. Sci. USA [ Proc. Nat. Acad. Sci. U.S. 77:5483 (1980); gilliland et al, proc. Nat ' l Acad. Sci. USA [ Acad. U.S. Sci ] 77:4539 (1980); krolick et al, proc. Nat ' l Acad. Sci. USA [ Acad. U.S. 77:5419 (1980)).

Other cytotoxic agents include the cytotoxic ribonucleases described by Goldenberg in U.S. patent No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates in which the radionuclide emitting alpha or beta particles is stably coupled to the antibody or binding fragment thereof, with or without the use of a complex forming agent. Such radionuclides include beta-emitters such as phosphorus-32 (³² P), scandium-47 (⁴⁷ Sc), copper-67 (⁶⁷ Cu), gallium-67 (⁶⁷ Ga), Yttrium-88 (⁸⁸ Y), yttrium-90 (⁹⁰ Y), iodine-125 (¹²⁵ I), iodine-131 (¹³¹ I), Samarium-153 (¹⁵³ Sm), lutetium-177 (¹⁷⁷ Lu), rhenium-186 (¹⁸⁶ Re) or rhenium-188 (¹⁸⁸ Re), and alpha-emitters, e.g., astatine-211 (²¹¹ At), Lead-212 (²¹² Pb), bismuth-212 (²¹² Bi) or-213 (²¹³ Bi) or actinium-225 (²²⁵ Ac).

Methods for conjugating antibodies or binding fragments thereof to detectable moieties and the like are known in the art, such as, for example, those described in Hunter et al Nature [ Nature ], 144:945 (1962); david et al Biochemistry [ Biochemistry ], 13:1014 (1974); pain et al J.Immunol. Meth. [ J.Immunol. Methods ], 40:219 (1981); and Nygren, J., histochem. And cytochem. [ histochemistry and cytochem ] 30:407 (1982).

The embodiments described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelid antibodies, nanobodies, igNAR, polypeptides, variable regions, and CDRs listed herein. These may contain, for example, conservative substitution mutations (i.e., substitution of one or more amino acids with similar amino acids). For example, conservative substitutions refer to the substitution of an amino acid with another amino acid in the same general class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another personality amino acid. The purpose of conservative amino acid substitutions is well known in the art.

In another embodiment, the invention contemplates polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of the antibody fragments, variable regions and CDRs listed herein. More preferably, the invention contemplates polypeptide sequences having at least 95% or more sequence homology, even more preferably at least 98% or more sequence homology, still more preferably at least 99% or more sequence homology to any one or more of the polypeptide sequences of the antibody fragments, variable regions and CDRs listed herein. Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

Another embodiment of the invention contemplates incorporating these polynucleotides into expression vectors for expression in mammalian cells such as CHO, NSO, HEK-293 or in fungal, insect or microbial systems such as yeast cells such as Pichia yeast. Suitable pichia species include, but are not limited to, pichia pastoris. In one embodiment of the invention described herein (below), fab fragments can be produced by enzymatic digestion (e.g., papain) of Ab6 after expression of the full length polynucleotide in a suitable host. In another embodiment of the invention, an anti-CGRP antibody such as Ab6 or Fab fragment thereof can be produced by expressing an Ab6 polynucleotide in mammalian cells such as CHO, NSO or HEK 293 cells, fungi, insect or microbial systems such as yeast cells (e.g., diploid yeast, e.g., pichia diploid) and other yeast strains. Suitable pichia species include, but are not limited to, pichia pastoris.

Host cells and vectors comprising the polynucleotides are also contemplated.

The invention further contemplates vectors comprising polynucleotide sequences encoding variable heavy and light chain polypeptide sequences as set forth herein, as well as respective complementarity determining regions (CDRs or hypervariable regions), as well as host cells comprising such vector sequences. In one embodiment of the invention, the host cell is a yeast cell. In another embodiment of the invention, the yeast host cell belongs to the genus pichia.

Methods of producing antibodies and fragments thereof

In another embodiment, the invention contemplates methods of producing anti-CGRP antibodies and fragments thereof. Methods for producing antibodies and fragments thereof secreted from polyploid (preferably diploid or tetraploid) strains of mating competent yeast are taught, for example, in U.S. patent application publication No. US 2009/0022659 to Olson et al and U.S. patent No. 7,935,340 to Garcia-Martinez et al, the disclosures of each of which are incorporated herein by reference in their entirety. Methods for producing antibodies and fragments thereof in mammalian cells, such as CHO cells, are well known in the art.

Other methods of producing antibodies are also known to those of ordinary skill in the art. For example, methods of producing chimeric antibodies are now well known in the art (see, e.g., U.S. Pat. No. 4,816,567 to cabily et al; morrison et al, P.N.A.S. USA [ Proc. Natl. Acad. Sci. USA ], 81:8651-55 (1984); neuberger, M.S. et al, nature [ Nature ] 314:268-270 (1985); boulianne, G.L. et al, nature [ Nature ] 312:643-46 (1984), the disclosure of each of which is incorporated herein by reference in its entirety).

Also, other methods of producing humanized antibodies are now well known in the art (see, e.g., U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 to Queen et al, U.S. Pat. Nos. 5,225,539 and 6,548,640 to winter, U.S. Pat. No. 6,054,297, 6,407,213 and 6,639,055 to Carter et al, U.S. Pat. No. 6,632,927 to Adair, jones, P.T. et al, nature [ Nature ], 321:522-525 (1986), reichmann, L. et al, nature [ Nature ], 332:323-327 (1988), verhoeyen, M et al, science [ Science ], 239:1534-36 (1988), the disclosures of each of which are incorporated herein by reference in their entirety).

Application of

"Pharmaceutical composition" refers to a chemical or biological composition suitable for administration to a mammal. Such compositions may be specifically formulated for administration by one or more of a variety of routes, including but not limited to intravenous or subcutaneous. Administration may be every 2 weeks, once a month (every 4 weeks), every other month, or every three months.

A "pharmaceutical excipient" or "pharmaceutically acceptable excipient" is a carrier, typically a liquid, in which the active therapeutic agent is formulated. In one embodiment of the invention, the active therapeutic agent is a humanized antibody or one or more fragments thereof as described herein. Excipients, while providing chemical and/or biological stability and release characteristics, generally do not provide any pharmacological activity to the formulation. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences [ rest pharmaceutical science ], 19 th edition, grennaro, a. Edit, 1995, which is incorporated by reference.

As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The present invention contemplates that the pharmaceutical composition exists in lyophilized form. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The present invention further contemplates the inclusion of a stabilizer in the pharmaceutical composition. For example, proper fluidity can be maintained in the case of dispersion by the maintenance of the desired particle size and by the use of surfactants.

In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, monostearates and gelatins. Moreover, the alkaline polypeptide may be formulated as a timed release formulation, for example as a composition comprising a slow release polymer. The active compounds can be prepared with carriers that will protect the compound from rapid release (e.g., controlled release formulations, including implants and microencapsulated delivery systems). Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid, polyglycolic acid copolymers (PLG) may be used. Many methods of preparing such formulations are known to those skilled in the art.

In accordance with the present invention, luAG09222 and eplerenone are co-formulated according to the following examples.

Unless otherwise indicated in the examples below, the total amount of epreneuzumab and LuAG09222 in the pharmaceutical formulation is between 100 mg/mL and 300 mg/mL (optionally about 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL or 300 mg/mL), wherein the ratio of epreneuzumab and LuAG00922 can vary. In one embodiment, the ratio of eplerenone and LuAG09222 is 1:1, 1:2, and 2:1, meaning that a ratio of, for example, 1:1 in 100 mg/mL pharmaceutical formulation will contain 50 mg/mL eplerenone and 50 mg/mL LuAG09222. Examples of pharmaceutical compositions according to the invention are given below.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 2:1, 1:1 or 1:2, and further comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, poloxamer P188.0025-0.0120% w/v and 30-70 mM NaCl, and having a pH of about 6 (optionally between pH 5.5 and 6.4, optionally pH 5.9).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, poloxamer P188 0.0025-0.010% w/v and 25-35 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 30 mM histidine buffer, 169 mM sorbitol, poloxamer P188 0.005% w/v and 30 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total combined protein concentration of eplerenone and LuAG09222 in the composition can be 113 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 30-50 mM histidine buffer, 80-150 mM sorbitol, L-arginine hydrochloride 150-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of eplerenone and LuAG09222 in the composition can be 140-150 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 30mM histidine buffer, 80 mM sorbitol, L-arginine hydrochloride 150 mM and poloxamer P188 0.02% w/v, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of eplerenone and LuAG09222 can be about 150 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 50 mM histidine buffer, 150 mM sorbitol, L-arginine hydrochloride 250 mM and poloxamer P188 0.02%, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of eplerenone and LuAG09222 can be about 145 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, poloxamer P188 0.005-0.010% w/v and 45-55 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 34 mM histidine buffer, 135 mM sorbitol, poloxamer P188 0.008% w/v and 48 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of eplerenone and LuAG09222 can be 120 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 30mM histidine buffer, 80 mM sorbitol, L-arginine 150 mM and poloxamer P188 0.02% w/v, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eplerenone and LuAG09222 can be about 130 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 10 mM histidine buffer, 10 mM sorbitol, L-arginine hydrochloride 50 mM and poloxamer P188 0.02% w/v, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eplerenone and LuAG09222 can be about 140 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:2 and comprising 35-45 mM histidine buffer, 90-100 mM sorbitol, poloxamer P188 0.010-0.0120% w/v and 65-75 mM NaCl and having a pH of about 6 (optionally pH 5.9).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 1:2, and comprising 38 mM histidine buffer, 96 mM sorbitol, poloxamer P188.0114% w/v and 69 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of eplerenone and LuAG09222 can be 129 mg/mL.

In the above embodiments, arginine (optionally L-arginine) may be added at a concentration of 10-150 mM, optionally 10mM, 50mM, 80mM, 100 mM or 120 mM or 150 mM.

Additionally, in embodiments including arginine, the pharmaceutical formulation may comprise eplerizumab and LuAG90222 in a ratio selected from 1:2, 1:1, or 2:1, and further comprise 30-50 mM histidine buffer, 50-250 mM sorbitol, and 10-150 mM NaCl, poloxamer 188 0.005-0.05% w/v, 10-150 mM L-arginine hydrochloride, and a pH of about 6 (optionally pH 5.9).

In another embodiment, the pharmaceutical formulation may comprise eplerizumab and LuAG90222 selected from 1:2, 1:1 or 2:1 and comprises 30-50 mM histidine buffer, 150-250 mM sorbitol and 80-150 mM NaCl, 0.005-0.05% w/v poloxamer P188, 80-150 mM L-arginine hydrochloride and has a pH of about 6 (optionally pH 5.9). The total protein concentration of eplerenone and LuAG09222 can be between 125-150 mg/mL.

In another embodiment, the pharmaceutical formulation may comprise eplerizumab and LuAG90222 in a ratio of 1:2, and comprises 10-50 mM histidine buffer, 50-250 mM sorbitol and 10-150 mM NaCl, 0.005-0.05% w/v poloxamer P188, 10-150 mM L-arginine hydrochloride, and has a pH of about 6 (optionally pH 5.9). The total protein concentration of eplerenone and LuAG09222 can be between 100-150 mg/mL.

In another embodiment, the pharmaceutical formulation may comprise eplerizumab and LuAG90222 in a ratio selected from 1:2, 2:1 and 1:1, and comprises 10-50 mM histidine buffer, 50-250 mM sorbitol and 10-150 mM NaCl, 0.005-0.05% w/v poloxamer P188, 10-150 mM L-arginine hydrochloride, and has a pH of about 7 (optionally pH 6.75) or a pH of about 5 (optionally about 5.25). The total protein concentration of eplerenone and LuAG09222 can be between 100-125 mg/mL.

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 1:1, 1:2 or 2:1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eplerenone and LuAG09222 can be between 140-155 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, or 155-mg/mL).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 1:1, 1:2 or 2:1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eplerenone and LuAG09222 can be between 140-155 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, or 155-mg/mL).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 1:1, 1:2 or 2:1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eplerenone and LuAG09222 can be between 140-155 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, or 155-mg/mL).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 1:1, 1:2 or 2:1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eplerenone and LuAG09222 can be between 140-160 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, 155-mg/mL, or 160-mg/mL).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio selected from 1:1 or 1:2, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eplerenone and LuAG09222 can be between 140-160 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, 155-mg/mL, or 160-mg/mL).

According to one embodiment, the present invention relates to a pharmaceutical composition comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 30-50 mM histidine buffer, 80-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and poloxamer P188 0.02% w/v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eplerenone and LuAG09222 can be between 140-160 mg/mL (optionally 140-mg/mL, 145-mg/mL, 150-mg/mL, 155-mg/mL).

Further exemplary embodiments

Example 1 (E1) a composition comprising eplerenone and LuAG09222.

E2. The composition of E1, further comprising histidine, poloxamer 188 or polysorbate 80.

E3. The composition according to E1 and E2, further comprising one, two or all of NaCl, sorbitol (e.g., L-sorbitol) and arginine (e.g., L-arginine).

E4. the pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG09222 in a total concentration of 100 mg/mL to 300 mg/mL.

E5. The pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG09222 in a total concentration of 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL or 300 mg/mL.

E6. The pharmaceutical composition of examples 4 or 5 having a ratio of eplerenone and LuAG09222 of 1:1, 1:2, or 2:1.

E7. the pharmaceutical composition according to any one of the preceding embodiments, wherein the composition comprises 100-300 mg/mL eplerenone and 50-100 mg/mL LuAG09222.

E8. The composition of any one of the preceding embodiments, wherein the composition comprises 100, 150, 200, or 300 mg/ml eplerizumab.

E9. The composition according to any one of the preceding embodiments, wherein the composition comprises 50, 75, 100 or 150 mg/ml LuAG09222.

E10. the composition of any one of the preceding embodiments, wherein the composition comprises 100 mg/ml eplerenone and 50 mg/ml LuAG09222.

E11. the composition of any one of the preceding embodiments, wherein eplerenone and LuAG09222 are the only active ingredients in the composition.

E12. the composition according to any one of the preceding embodiments, wherein histidine is in the composition at a concentration of between 10-50 mM, optionally 10 mM, 20 mM, 25 mM, 30 mM, 40 mM or 50 mM.

E13. The composition of any one of the preceding embodiments, wherein polysorbate 80 or poloxamer P188 is 0.005-0.05% w/v in the composition.

E14. The composition according to any one of the preceding embodiments, wherein one, two or three of the following excipients are present in the composition, namely NaCl, sorbitol and arginine.

E15. the composition according to any one of the preceding embodiments, wherein the concentration of NaCl in the composition is between 10-150 mM, optionally 25 mM, 50mM, 100 mM or 150 mM.

E16. the composition according to any one of the preceding embodiments, wherein sorbitol is present in the composition at a concentration of between 50-250 mM, optionally 50mM, 100 mM, 150 mM, 200 mM or 250 mM.

E17. The composition of any one of the preceding embodiments, wherein arginine is present in the composition at a concentration of between 50-250 mM, optionally 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM.

E18. The composition of any of the preceding embodiments, wherein the pH is between 5.0-6.8, optionally pH 5.0, pH 5.5, pH 5.9, pH 6.0, pH 6.5, or pH 6.8.

E19. The pharmaceutical composition according to any of the preceding claims, wherein the concentration of histidine ranges between 10-50 mM, optionally 20-40 mM, the concentration of poloxamer P188 ranges between 0.0025-0.0120% w/v, and the concentration of polysorbate 80 ranges between 0.005-0.05% w/v, inclusive.

E20. The pharmaceutical composition according to any of the preceding embodiments, wherein the concentration of NaCl is between 10-150 mM, optionally 30-70 mM, the concentration of sorbitol is between 50-250 mM, optionally 90-180 mM, and the concentration of arginine (e.g. L-arginine) is between 50-250 mM, inclusive.

E21. the pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG90222 in a ratio of 2:1, 1:1 or 1:2, and comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, poloxamer P188 0.0025-0.0120% w/v and 30-70 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

E22. The pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG90222 in a ratio of 2:1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, poloxamer P188 0.0025-0.010% w/v and 25-35 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

E23. The pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG90222 in a ratio of 1:1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, poloxamer P188 0.005-0.010% w/v and 45-55 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

E24. the pharmaceutical composition according to any one of the preceding embodiments, comprising eplerenone and LuAG90222 in a ratio of 1:2, and comprising 35-45 mM histidine buffer, 90-100 mM sorbitol, poloxamer P188 0.010-0.0120% w/v and 65-75 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

E25. the composition according to any one of the preceding embodiments for use as a medicament.

E26. The composition of any one of the preceding embodiments for use in subcutaneous administration.

E27. The composition according to any one of the preceding embodiments for use in treating or preventing headache.

E28. The composition according to any one of the preceding embodiments for use in the treatment or prevention of pain.

E29. the composition according to any one of the preceding embodiments for use in the treatment or prevention of chronic or episodic migraine.

E30. The composition according to any one of the preceding embodiments for use in the treatment or prevention of cluster headache.

E31. the composition according to any one of the preceding embodiments for use in the treatment or prevention of endometriosis.

E32. The composition according to any one of the preceding embodiments for use in the treatment or prevention of: migraine (with or without aura), weight loss, cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, hemiplegic migraine, cluster headache, migraine neuralgia, chronic headache, tension headache, general headache, hot flushes, chronic onset migraine, secondary headache caused by potential structural problems of the head or neck, cerebral neuralgia, sinus headache (optionally such as sinusitis related headache), allergy-induced headache or migraine, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, bone fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with: gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menopause, childbirth, menopause, prostatitis or pancreatitis.

E33. The composition according to any one of the preceding embodiments for use in the treatment or prophylaxis of chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, ocular pain, dental pain, post-operative pain, wound-related pain, diabetes, non-insulin dependent diabetes mellitus and other inflammatory autoimmune disorders, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hyperreactivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flushes in men and women, allergic dermatitis, psoriasis, encephalitis, brain trauma, epilepsy, neurodegenerative diseases, skin disorders including itch, neuropathic skin redness, skin roses and erythema, inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea.

E34. The composition according to any of the preceding embodiments, for administration monthly or every 2 weeks.

E35. the composition of any one of the preceding embodiments, wherein the composition comprises eplerenone and fragments of LuAG 09222.

E36. the composition of example 26, wherein the fragments of eplerenone and LuAG09222 comprise all 6 CDR regions of the antibody, respectively.

E37. the composition of embodiment 26, wherein the fragment comprises or consists of VH and VL regions of eplerenone and LuAG09222.

E38. The composition of any one of the preceding embodiments, wherein the composition comprises 50-150 mg/ml eplerenone, optionally 50 mg/ml, 75 mg/ml, 100 mg/ml, or 150 mg/ml.

E39. The composition according to any one of the preceding embodiments, wherein the composition comprises 25-100 mg/ml LuAG09222, optionally 25 mg/ml, 50 mg/ml, 75 mg/ml, or 100 mg/ml.

E40. the composition according to any of the preceding embodiments for use in a method of subcutaneous administration.

E41. a method for treating or preventing headache, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

E42. A method for treating or preventing pain, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

E43. A method for treating or preventing chronic or episodic migraine, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

E44. A method for treating or preventing cluster headache, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

E45. a method for treating or preventing endometriosis, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

E46. A method for treating or preventing: migraine (with or without aura), weight loss, cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, hemiplegic migraine, cluster headache, migraine neuralgia, chronic headache, tension headache, general headache, hot flushes, chronic onset migraine, secondary headache caused by potential structural problems of the head or neck, cerebral neuralgia, sinus headache (optionally such as sinusitis related headache), allergy-induced headache or migraine, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, bone fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with: gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menopause, childbirth, menopause, prostatitis or pancreatitis,

The method comprises administering to a patient in need thereof a composition according to any one of E1-E40.

E47. A method for treating or preventing chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, ocular pain, dental pain, postoperative pain, trauma related pain, diabetes, non-insulin dependent diabetes mellitus and other inflammatory autoimmune disorders, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hyperreactivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flushes in men and women, allergic dermatitis, psoriasis, encephalitis, brain trauma, epilepsy, neurodegenerative diseases, skin diseases including itch, neurogenic skin redness, skin roses and erythema, inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea,

The method comprises administering to a patient in need thereof a composition according to any one of E1-40.

E48. use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of headache.

E49. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of pain.

E50. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of chronic or episodic migraine.

E51. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of cluster headache.

E52. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of endometriosis.

E53. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prophylaxis of migraine (with or without aura), weight loss, cancer or tumour, angiogenesis associated with cancer or tumour growth, angiogenesis associated with cancer or tumour survival, hemiplegic migraine, cluster headache, migraine neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic onset migraine, secondary headache due to head or neck underlying structural problems, cerebral neuralgia, sinus headache (optionally e.g. sinusitis related headache), allergy-induced headache or migraine, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, bone fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crises and other nociceptive pain, and hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with: gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menopause, childbirth, menopause, prostatitis or pancreatitis.

E54. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prophylaxis of chronic pain, neurogenic and inflammatory pain, neuropathic pain, ocular pain, dental pain, postoperative pain, wound-related pain, diabetes mellitus, non-insulin dependent diabetes mellitus and other inflammatory autoimmune disorders, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hyperreactivity, asthma, shock, sepsis, withdrawal syndrome, morphine tolerance, hot flushes in men and women, allergic dermatitis, psoriasis, encephalitis, brain trauma, epilepsy, neurodegenerative diseases, skin disorders including pruritus, neurogenic skin redness, skin roses and erythema, inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea.

E55. The composition, method or use of any one of the preceding embodiments, wherein eplerenone is administered with a single antibody comprising all six CDR sequences:

Light chain CDR1 SEQ ID NO:7, light chain CDR2 SEQ ID NO:8, light chain CDR3 SEQ ID NO:9, heavy chain CDR1 SEQ ID NO:1, heavy chain CDR2 SEQ ID NO:2, and heavy chain CDR3 SEQ ID NO:3.

E56. the composition, method or use according to any of the preceding embodiments, wherein LU AG 09222 comprises all six CDR sequences:

Light chain CDR 1 SEQ ID NO:17, light chain CDR 2 SEQ ID NO:18, light chain CDR 3 SEQ ID NO:19, heavy chain CDR 1 SEQ ID NO:12, heavy chain CDR 2 SEQ ID NO:13, and heavy chain CDR 3 SEQ ID NO:14.

E57. The composition, method or use of any of the preceding embodiments, wherein eplerenone has a VH region as defined in SEQ ID No.: 4 and a VL as defined in SEQ ID No.: 10.

E58. the composition, method or use according to any of the preceding embodiments, wherein LU AG09222 has a VH region as defined in SEQ ID No.: 15 and a VL region as defined in SEQ ID No.: 20.

E59. The composition, method or use according to any one of the preceding embodiments, wherein the eplerenone is a monoclonal antibody having a heavy chain as defined in SEQ ID No. 5 or SEQ ID No. 6 and a light chain as defined in SEQ ID No. 11.

E60. The composition, method or use according to any of the preceding embodiments, wherein the LU AG09222 has a heavy chain as defined in SEQ ID No.: 16 or SEQ ID No.: 26 and a light chain as defined in SEQ ID No.: 21.

E61. The composition, method or use of any of the preceding embodiments, wherein histidine is in the form of L-histidine and/or sorbitol is in the form of L-sorbitol and arginine is in the form of L-arginine.

Further Exemplary Embodiment (EE)

Example 1 (EE 1) a method for treating a patient in need thereof by administering effective amounts of eplerenone and LuAG 09222.

Ee2. the method according to EE1, wherein eplerenone is administered simultaneously or sequentially with LuAG 09222.

Ee3. the method according to any one of the preceding embodiments, wherein eplerenone and LuAG09222 are administered sequentially over a period of 1 ⁄ -2 hours, e.g., 1 ⁄ hours or 1 hour.

Ee4. the method according to any one of the preceding embodiments, wherein 50-150 mg/ml eplerenone is administered, optionally 50 mg/ml, 75 mg/ml, 100 mg/ml, or 150 mg/ml.

Ee5. the method according to any one of the preceding embodiments, wherein 25-100 mg/ml LuAG09222, optionally 25 mg/ml, 50 mg/ml, 75 mg/ml or 100 mg/ml is administered.

Ee6. the method according to any one of the preceding embodiments, wherein eplerenone is administered subcutaneously or intravenously and LuAG09222 is administered subcutaneously or intravenously.

Ee7. the method according to any one of the preceding embodiments, for treating or preventing headache.

Ee8. the method according to any one of the preceding embodiments, for treating or preventing pain.

Ee9. the method according to any one of the preceding embodiments, for treating or preventing chronic or episodic migraine.

Ee10. the method according to any one of the preceding embodiments, for treating or preventing cluster headache.

EE11 according to the method of any one of the preceding embodiments, for the treatment or prevention of endometriosis.

Ee12 the method according to any of the preceding embodiments for treating or preventing migraine (with or without aura), weight loss, cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, hemiplegic migraine, cluster headache, migraine neuralgia, chronic headache, tension headache, general headache, hot flushes, chronic onset migraine, secondary headache caused by potential structural problems of the head or neck, cerebral neuralgia, sinus headache (e.g. such as sinusitis related headache), allergy-induced headache or migraine, pain, inflammatory pain, post-operative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, bone fracture pain, osteoporosis fracture pain, pain resulting from burns, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, liver cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with: gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menopause, childbirth, menopause, prostatitis or pancreatitis.

Ee13 the method according to any one of the preceding embodiments for treating or preventing chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, ocular pain, dental pain, post-operative pain, wound-related pain, diabetes, non-insulin dependent diabetes mellitus and other inflammatory autoimmune disorders, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hyperreactivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flushes in men and women, allergic dermatitis, psoriasis, encephalitis, brain trauma, epilepsy, neurodegenerative diseases, skin diseases including itch, neurogenic skin redness, skin rose-like spots and erythema, inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea.

Ee14. the method according to any one of the preceding embodiments, wherein eplerenone and LuAG09222 are administered monthly (every 4 weeks) or every 2 weeks.

Ee15. the method according to any one of the preceding embodiments, wherein ependelizumab comprises all six CDR sequences:

Light chain CDR1 SEQ ID NO:7, light chain CDR2 SEQ ID NO:8, light chain CDR3 SEQ ID NO:9, heavy chain CDR1 SEQ ID NO:1, heavy chain CDR2 SEQ ID NO:2, and heavy chain CDR3 SEQ ID NO:3.

EE16 the method according to any one of the preceding embodiments, wherein LUAG09222 comprises all six CDR sequences:

Light chain CDR 1 SEQ ID NO:17, light chain CDR 2 SEQ ID NO:18, light chain CDR 3 SEQ ID NO:19, heavy chain CDR 1 SEQ ID NO:12, heavy chain CDR 2 SEQ ID NO:13, and heavy chain CDR 3 SEQ ID NO:14.

EE17 the method according to any one of the preceding embodiments, wherein eplerenone has a VH region as defined in SEQ ID No.: 4 and a VL as defined in SEQ ID No.: 10.

EE18 the method according to any one of the preceding embodiments, wherein LU AG09222 has a VH region as defined in SEQ ID No.: 15 and a VL region as defined in SEQ ID No.: 20.

EE19 the method according to any one of the preceding examples, wherein the eplerenone is having a heavy chain as defined in SEQ ID No.: 5 or SEQ ID No.: 6 and a light chain as defined in SEQ ID No.: 11.

The method according to any of the preceding embodiments, wherein the LU AG09222 has a heavy chain as defined in SEQ ID No.: 16 or SEQ ID No.: 26 and a light chain as defined in SEQ ID No.: 21.

The above description of various illustrative embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed. Although specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. In addition to the examples described above, the teachings of the present invention provided herein may be applied to other purposes.

These and other changes can be made to the invention in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification and the claims. Accordingly, the invention is not limited by this disclosure, but rather the scope of the invention is to be determined entirely by the following claims.

The invention may be practiced otherwise than as specifically described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) cited in the background, detailed description, and examples of the invention is hereby incorporated by reference in its entirety.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what is claimed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, concentration, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure.

Examples

The following examples are provided to illustrate the invention and should not be construed as limiting the scope of the claims in any way.

Example 1

Human clinical study to evaluate the safety and efficacy of anti-CGRP antibodies according to the invention

Clinical treatment protocol

The ability of humanized anti-CGRP IgG1 antibodies identified herein as Ab6 (eplercanizumab) to inhibit, reduce, or prevent the number, duration, and/or intensity of migraine attacks was evaluated in human subjects.

In particular, the clinical efficacy of Ab6 antibodies was tested in a placebo-controlled double-blind randomized study. All individuals in the study were selected based on specific criteria. In particular all patients diagnosed with migraine (age. Ltoreq.50 years) (ICHD-II, section 1 of 2004) and having a history of migraine of. Gtoreq.12 months, with. Gtoreq.5 and. Ltoreq.14 migraine days every 28-day period within 3 months prior to screening.

In addition, all individuals in the study used acute migraine medications for < 14 days every 28-day period and triptans for < 10 days every 28-day period during the 28-day period in the 3 months prior to screening and in the 28-day period when the electronic diary was completed prior to randomization.

Table 1 summarizes the demographic characteristics of the study population.

All individuals had to record their migraine conditions daily throughout the study using an electronic diary (e-diary). In the electronic diary, subjects in the study were required to record migraine days/month, migraine episodes/month, migraine hours/month, migraine severity, and any abortive medications (e.g., triptans) use.

Furthermore, study participants were required to record their migraine conditions using an electronic diary over a 28 day period prior to treatment with antibodies or placebo to establish a monthly migraine day/hour/episode baseline. Furthermore, this familiarizes the subject under study with the use of electronic diaries.

After 28 days of break-in, subjects in the study were divided into two groups, each group comprising 80 subjects (fig. 5). In the first group, the antibody-treated group (n=80), each subject in the group was administered a single 1000 mg dose of Ab6 intravenously. In the second group (n=80), placebo group, each subject was given an intravenous injection containing only an aqueous antibody carrier solution.

Individuals in the treatment group and placebo group were evaluated in 24 weeks after dose administration. Initially, a 12-cycle period analysis was performed. After the 12-cycle period analysis, a fine analysis was performed. Such fine analysis may include, for example, adding or deleting patient data according to a study protocol, e.g., updating data that has not been fully loaded from an electronic diary. This refinement resulted in slight variations, but did not change the overall conclusion.

The efficacy of the antibodies compared to placebo was evaluated based in part on the data recorded in the electronic diary entry. For example, the analysis includes comparing the recorded migraine day/month, migraine attacks/month, and migraine hours/month of the subject between the treatment group compared to the placebo group. The percentage of respondents (i.e., subjects with 50%, 75% and 100% decrease in migraine days) in each of the two groups were also compared.

In addition, the response of Ab6 treated and placebo treated subjects to MSQ and HIT-6 questionnaires in both groups will be evaluated and compared. MSQ is a commonly used disease-specific tool for assessing the impact of migraine on health-related quality of life (HRQL). MSQ contained 16 migraine specific quality of life questionnaires (version 1.0) developed by Glaxo wellcom inc. It is assumed that MSQ can measure 3 parameters, (i) role function-restriction, (ii) role function-prophylactics, and (iii) mood function.

HIT-6 or functional impact (also known as headache impact test or HIT-6) is similarly a well-known tool for assessing migraine intensity. Six questions were used to illustrate the effect of headache and its treatment on functional health and well-being of an individual.

Clinical results and analysis

The results of the human clinical trial and analysis by week 12 in the treated subjects are summarized in table 2 below.

TABLE 2 respondent analysis of migraine day

Furthermore, clinical study results were compared based on the number of responders in the treatment group and placebo group. As shown in fig. 1, the reduction in migraine days per month in the mid-term was compared by 50%, 75% or 100% in the number of subjects in the treatment group and the placebo group. As shown, the headache day was reduced by at least 50% for 60% of subjects in the Ab6 treated group, by at least 75% for 31% of subjects in the Ab6 treated group, and by 100% for 15% of subjects in the Ab6 treated group.

In contrast, the headache days were reduced by at least 50% for 33% of the subjects in the placebo-treated group, by at least 75% for 9% of the subjects in the placebo-treated group, and 100% for 0% (none) of the subjects in the placebo-treated group.

These results clearly demonstrate that the migraine days for Ab6 treated group were reduced more. But the differences between these numbers will be more pronounced for a significant placebo effect. (the effect of elevated placebo is not surprising, as this is generally high for migraine and other neurological drugs).

In addition, the placebo and Ab6 treated groups were compared for% change from baseline in the number of migraine days per month. As shown in fig. 2, the median (±qr) change% from baseline for the monthly migraine days between 12 post-treatment periods was compared for the 2 groups of placebo and Ab6 treated groups. These results with statistical significance (p=0.0078) clearly show that Ab6 treated group had a greater reduction in monthly headache days than placebo treated group compared to baseline.

In addition, the placebo and Ab6 treated groups were compared for% change in the number of migraine episodes per month from baseline. As shown in fig. 3, the median (±qr) change% from baseline in the number of migraine episodes per month between 12 post-treatment periods was compared for the placebo and Ab6 treated groups. These results demonstrate that Ab6 treated group had significantly more reduction in the number of migraine episodes per month than placebo treated group compared to baseline.

In addition, the placebo and Ab6 treated groups were compared for% change from baseline in the number of migraine hours per month. As shown in fig. 4, the median (±qr) change% from baseline was compared for the 2 groups of placebo and Ab6 treated groups for the number of migraine hours per month between 12 post-treatment periods. These results clearly demonstrate that Ab6 treated group had a greater reduction in the number of migraine hours per month compared to baseline compared to placebo treated group.

In addition, the HIT-6 results of the two groups were compared. As previously mentioned, the questionnaire is widely accepted for use in assessing migraine conditions in frequent/chronic migraine individuals. Figure 6 compares HIT-6 responder analysis for Ab6 treated group and placebo group at baseline, week 4 post-treatment, week 8 post-treatment, and week 12 post-treatment. The results at each time point showed a statistically significant improvement in HIT-6 score for the Ab6 treated group compared to the placebo group, i.e., 54.4% versus 30% of the Ab6 treated group at week 4 (p=0.0023), 51.3% versus 38.0% of the Ab6 treated group at week 8 (p= 0.1094) and 61.1% versus 33.3% of the Ab6 treated group at week 12 (p=0.0007). Figure 7 shows the percentage of patients with some or little/no HIG-6 scores over time in placebo and Ab6 treated groups (showing statistical significance).

In addition, figure 8 contains the Pharmacokinetic (PK) profile of Ab6 administered intravenously at a single dose of 1000 mg (mg/mL) over a 24 week period following Ab6 administration.

Figure 9 includes plasma free Pharmacokinetic (PK) parameters N (patient number), mean and Standard Deviation (SD) for Ab6 at a single intravenous dose of 1000 mg. The parameters and units shown in the tables are C _max（µg/mL）、AUC_0-∞ (mgHr/mL), half-life (days), V _z (L), and C _L (mL/hr).

Patient data were further analyzed from 12 weeks to 24 weeks. The treated group continued to exhibit reduced migraine days compared to the control group, but the magnitude of the difference decreased over time. In addition, the control group showed less migraine day per month than baseline. This is believed to be due, at least in part, to "diary fatigue" in which case the patient may not report migraine on the day when migraine actually occurred, avoiding spending time and effort answering more questions about migraine because they give a positive answer to the question of whether migraine is present on a certain day.

Further analysis of the results of the study are shown in figures 10 to 21. These results included analysis of changes from baseline (mean +/-SEM) in Ab6 (1000 mg i.v.) versus placebo monthly migraine day (fig. 22), mean migraine day over time (+/-SD) for the entire analysis population (fig. 23). In addition, the distribution and variation of the actual migraine days of the Ab6 treated group during weeks 1-4 (fig. 12), the distribution and variation of the actual migraine days of the placebo group during weeks 1-4 (fig. 13), the distribution and variation of the actual migraine days of the Ab6 treated group during weeks 5-8 (fig. 14), the distribution and variation of the actual migraine days of the placebo group during weeks 5-8 (fig. 15), the distribution and variation of the actual migraine days of the Ab6 treated group during weeks 9-12 (fig. 16), and the distribution and variation of the actual migraine days of the placebo group during weeks 9-12 (fig. 17) are shown.

Responder rate analysis was also performed (fig. 18-20). These figures show 50%, 75% and 100% responder rates for Ab6 and placebo treated groups, respectively. Subjects with a migraine frequency decrease of 50% or more were considered 50% responders. Subjects with a migraine frequency decrease of > 75% were considered 75% responders. Likewise, subjects with 100% decrease in migraine frequency were considered 100% responders.

In fig. 10 and 18 to 20, the visit interval (in which the electronic diary was completed for 21-27 days) was normalized by multiplying the observed frequency by the reciprocal of the completion rate.

Migraine severity was also analyzed. Figure 21 shows the average migraine severity over time for the whole analysis population. On the scale used, an average score of 3 for migraine pain represents "moderate pain".

Figure 22 summarizes the changes in migraine day, migraine attacks, migraine hours, average migraine severity, headache frequency, and outcome measure (including HIT-6 score, MSQ (migraine specific quality of life questionnaire) RFP (role function-preventative), MSQ RFR (role function-restriction), and MSQ EF (mood function)) from baseline.

Example 2

Human clinical study to assess safety and efficacy of anti-CGRP antibodies in chronic migraine patients

This example describes a randomized, double-blind, placebo-controlled clinical trial to evaluate the safety and efficacy of Ab6 (eplerizumab) in preventing chronic migraine. In this study 1,072 patients were randomized to receive Ab6 (300 mg or 100 mg) or placebo administered by infusion once every 12 weeks. To qualify for the trial, the patient must go through at least 15 headache days per month, at least eight of which meet the migraine standard. Patients enrolled in this trial had an average of 16.1 migraine days per month at baseline. Study endpoints included average changes from baseline in monthly migraine days, reduced migraine prevalence on days 1 and 1-28, and at least 50%, 75% and 100% decrease from baseline in average monthly migraine days, changes from baseline in average monthly acute migraine specific dosing days, reduced impact scores from baseline reported by patients of headache impact test (HIT-6).

Patient characteristics are summarized in fig. 27, where the separate columns are for patients receiving placebo, 100 mg antibody, or 300 mg antibody. The average years after diagnosis of migraine in patients is 17.0 to 19.0 years, the average duration of chronic migraine is 11.5 to 12.4 years, and the patients with at least one prophylactic agent are 44.3% to 45.2%. At baseline, the average migraine days per month for the two antibody-treated groups was 16.1, while the average migraine days per month for the placebo group was 16.2.

The average monthly migraine days reduced by a specified percentage (50%, 75% or 100%) from baseline refers to the number or percentage of patients in the treatment group that exhibit a given percentage reduction in the monthly migraine days. For example, if the number of migraine days per month decreases by at least 12 days per month over a specified period of time, patients exhibiting a 16-month migraine day at baseline would be 75% respondents.

The results are shown in fig. 23 to 27. Figure 23 shows the percentage of migraine sufferers in the 300 mg, 100 mg and placebo treated groups on days 1, 7, 14, 21 and 28. The top row shows placebo results, the bottom row shows 300 mg doses and the middle row shows 100 mg doses.

As shown in fig. 23, on day 1, the percentage reduction in migraine prevalence was 52% at 300 mg doses, 50% at 100 mg doses, and 27% placebo. The decrease shown was statistically significant in both the 100 mg and 300 mg treated groups compared to the placebo group.

Figures 24-26 show the percentage of patients in the 300 mg and 100 mg treatment groups who reached 50%, 75% and 100% reduction in migraine day in month 1, month 1-3 (after infusion 1) and month 4-5 (after infusion 2), respectively. In each figure, the data bars show the results of 100 mg, 300 mg and placebo groups from left to right. Statistical significance is shown below. ++ means statistically significant difference from placebo; + represents statistically significant differences from placebo (unregulated), and ≡represents statistically significant differences from placebo (post-analysis).

Example 3

Baseline subgroup analysis of human clinical studies for assessing safety and efficacy of anti-CGRP antibodies in chronic or episodic migraine patients

In the chronic migraine study described in example 3, each patient was evaluated for potential drug overdose headache (MOH) at the time of ingestion. MOH appeared in 39.9% (139 patients) in the 100 mg treated group, 42.0% (147 patients) in the 300 mg treated group, and 39.6% (145 patients) in the placebo group. Evaluation of the treatment results in this patient subgroup indicated that treatment with anti-CGRP antibodies was effective for MOH (fig. 29). Specifically, in the 100 mg treatment group, the average monthly migraine day of patients with MOH at baseline was changed by-3.0 days (95% CI, -4.56 to-1.52 days) compared to the MOH patients receiving placebo. Similarly, in the 300 mg treatment group, the average monthly migraine day for patients with MOH at baseline was changed by-3.2 days (95% CI, -4.66 to-1.78 days) compared to the MOH patients receiving placebo. In contrast, for patients without MOH at baseline, the average monthly migraine day was changed by-1.3 days (95% CI, -2.43 to-0.16 days) in the 100 mg treatment group compared to the patients without MOH at baseline who received placebo. Also, for patients without MOH at baseline, the average monthly migraine day was changed by-2.1 days (95% CI, -3.24 to-0.88 days) in the 300 mg treatment group compared to the patients without MOH at baseline who received placebo. Efficacy is also shown for other subgroups including patients with average migraine day (MMD) frequency of less than 17 days or greater than 17 days, patients with age less than or equal to 21 years or greater than 21 years at diagnosis, patients with migraine duration less than or equal to 15 years or greater than 15 years, patients with migraine aura or without aura, patients with or without prior preventative medication, patients with or without concomitant preventative medication, and patients with or without a number of days greater than or equal to 33% of triptans or less than 33% of days. In each case, the efficacy of each subgroup is shown (fig. 29).

In a human clinical trial with another patient with episodic migraine, in a double-blind, parallel study, the patient received Ab6 100 mg (n=221), 300 mg (n=222) or placebo (n=222) at random. After a 28-day screening period, patients were given either drug or placebo intravenously every 3 months for a total of 4 infusions (fig. 28). Efficacy was shown at months 1-3 for both the 100 mg and 300 mg treated groups, with an average change in migraine daily of-3.9 days for the 100 mg treated group and-4.3 days for the 300 mg treated group compared to-3.2 days for the placebo group. Also shown are efficacy for a subset of patients, including those with average migraine daily (MMD) frequency of less than or equal to 9 days or greater, those with age less than or equal to 21 years or greater at diagnosis, those with migraine duration less than or equal to 15 years or greater, and those with or without migraine aura.

Example 4

Ab6 treatment effects on chronic and episodic migraine patients.

In the study of chronic migraine sufferers described in example 3 and episodic migraine sufferers described in example 4, the sufferers also recorded the use of acute medications in a daily electronic diary and allowed them to decide on the use of acute medications. Acute medications for migraine include ergots, triptans, and analgesics (e.g., NSAIDs, opioids, and caffeine-containing combination analgesics).

For further analysis, patients were stratified for the days of acute drug use during the 28-day screening period (1-9 or ≡10 days; "baseline"). Acute medication days for each type of acute medication and combination (meaning that if 2 or more types of medications are used on the same calendar day, they are counted as individual medication use days) were calculated. For example, if the patient took opioids and triptans on the same day, they are considered to be acute for 2 days. These assays included patients with at least 1 acute dosing day during the 28-day baseline screening period.

In chronic migraine and narcotic migraine patients with acute drug during the 28 day baseline period, ab6 treatment reduced the average monthly migraine day and acute drug day by greater than placebo at the earliest month 1 after administration, with similar results spanning 2 dose intervals over 6 months.

In chronic migraine patients with acute drug ≡1 day during baseline, ab6 continued to show a greater decrease in average monthly migraine days over placebo in 6 months of treatment (figure 30). Chronic migraine patients who had acute drug use at least one day per month during baseline showed a greater reduction in acute drug use than placebo as early as month 1 after treatment and during the entire 6 month treatment period (figure 31). In a subset of chronic migraine patients taking 1-9 days of acute drug during baseline, 300 mg of acute drug of the Ab6 group was used for a treatment period of 6 months with a greater change in days of acute drug use than placebo compared to baseline (figure 32). A significant reduction in the number of days of drug use per month was observed for patients who used drug at least 10 days per month at baseline for both Ab6 treatment groups compared to placebo throughout the 6 month period. FIG. 33 shows the change in days of drug use at month 1 and month 6 in a subgroup of chronic migraine patients who used acute drugs at baseline for 1 day, 1-9 days, and 10 days. Ab6 showed greater therapeutic effect than placebo in reducing acute drug use, except for 100 mg Ab6 at month 6 for patients on days 1-9/month of baseline use.

Similarly, episodic migraine patients with acute drug used for one or more days during baseline experienced a greater reduction in average monthly migraine days with Ab6 than placebo, spanning 2 dose intervals over 6 months (fig. 34). Patients with episodic migraine who had acute drug use at least one day per month during baseline showed a greater reduction in acute drug use than placebo as early as the 1 st month after treatment and during the entire 6 month treatment period (fig. 35). In the subset of narcotic migraine patients taking 1-9 days of acute drug during baseline, the number of days of acute drug use with Ab6 was greater than the placebo during the 6 month treatment period (fig. 36). Similar patterns were observed in subgroups of patients taking acute drugs for > 10 days during baseline, although smaller sample volumes may lead to less consistent patterns over time. FIG. 37 shows the change in days of drug use at month 1 and month 6 in a subset of narcotic migraine patients using acute drugs at baseline for 1 day, 1-9 days, and 10 days. Patients with a baseline usage of ≡10 days/month reduced acute drug usage of the Ab6 treated group by more than placebo, except for Ab 6100 mg at month 6.

The results show that both narcotic migraine and chronic migraine patients, who are at risk of drug overuse headache (use of acute drug ≡10 days/month), show the greatest decrease in acute drug use, where Ab6 treatment generally results in a greater decrease in drug use days than placebo.

In > 10% of subjects, the most commonly reported acute headache medications include Thomapyrin N (44.5%) (combination of acetaminophen, aspirin, and caffeine), ibuprofen (40.6%), sumatriptan (33.6%), acetaminophen (20.3%), and naproxen sodium (10.2%). In > 10% of subjects, the most commonly reported prophylactic headache drug is topiramate (12.5%).

Example 5

Efficacy of anti-CGRP antibodies in subjects experiencing migraine episodes

This example describes a randomized, double-blind, placebo-controlled clinical trial to evaluate the safety and efficacy of Ab6 for acute migraine treatment. In this study, approximately 450 patients received 100 mg of Ab6 or placebo at 1:1 ratio at random. During the screening period (about 1-8 weeks), patients were assessed for migraine frequency and drug use frequency. Eligible patients had a frequency of migraine attacks of about 4-15 migraine days per month within 3 months prior to screening. Historically, if left untreated, a typical migraine attack in a subject will be associated with moderate to severe headache and the most disturbing symptoms of nausea, photophobia or voice intolerance. The subject must be free of headache for at least 24 hours before a qualified migraine attack can participate in the trial. On the day of treatment, patients will go to the study site and begin intravenous infusion of 100 mg of Ab6 or placebo about 1-6 hours from onset. The patient will not receive any other monoclonal antibodies (e.g., any CGRP antagonist antibodies) for a period of 6 months prior to screening.

The primary endpoint was time to no headache and time without the most disturbing symptoms. The key secondary endpoint was no headache at 2 hours and no most disturbing symptoms at 2 hours. The secondary endpoints were time to headache relief, no headache at 2 hours for 24 and 48 hours without headache, use of rescue medication before 24 hours and before 48 hours, no photophobia at 2 hours, no nausea at 2 hours, change in headache impact test at week 4 (HIT 6) from baseline, and change in migraine treatment optimization questionnaire 6 at week 4 (mTOQ-6) from baseline. Exploratory endpoints were no headache at all time points except 2 hours, no photophobia at all time points except 2 hours, no nausea at all time points except 2 hours, no pain recurrence when the subject was headache free at 2 hours, patient global change impression (PGIC) at week 4 and time to next migraine. Headache was collected on a 4-point scale, with 3 being severe, 2 being moderate, 1 being mild, and 0 being pain-free. In the absence of rescue medication, no pain was pain (0) (note that in the test rescue medication must not be used within 2 hours after infusion was completed to distinguish the effect of the antibody from rescue medication, but during normal use rescue medication may optionally be used; the use of any rescue medication is collected as data).

Statistical analysis was performed to determine the significance of endpoint differences between patients receiving Ab6 or placebo, including time to no pain and time to no most disturbing symptoms, as well as each of the other endpoints described above.

By rescue medication is meant any intervention (medical or device) provided to a subject to alleviate migraine. In this study, rescue medication should not be provided within 2 hours after completion of study medication administration to distinguish the effect of the antibody from that of the rescue medication, but use of rescue medication is not prohibited. The study summarises the proportion of subjects in need of rescue medication. Acute rescue drugs include any drug that treats migraine or symptoms associated with migraine, such as triptans, analgesics (e.g., non-opioids or opioids/anesthetics), acetaminophen, NSAIDs, combination drugs (e.g., EXCEDRIN ^® or EXCEDRIN MIGRAINE ^®), antiemetics, ergotamines, ergotamine derivatives, and the like.

As reported by the subject, the absence of migraine related symptoms (photophobia, photophobia and nausea) refers to the absence or presence of the above migraine related symptoms. The study summarises the proportion of subjects who were asymptomatic in the absence of administration of rescue medication.

Headache impact test (HIT-6) was evaluated as the change in total score versus baseline and summarized in the study and compared between treatment groups.

Migraine treatment optimization questionnaire 6 (mTOQ-6) was evaluated as the change in total score versus baseline and differences between treatment groups were summarized and compared in the study.

The time to headache relief is assessed as the first time point after infusion completion at which subjects reported pain relief, meaning that their headache had changed from moderate or severe (2 or 3) to mild or no pain (1 or 0) without administration of rescue medication.

Pain recurrence was assessed as headache of any severity occurring within 48 hours of drug administration for patients without headache (0) at 2 hours. The study summarises the proportion of headache-relapsing patients of any severity.

This study showed that Ab6 was effective and safe for acute migraine treatment.

Example 6

In a critical clinical study, patients received 100 mg or 300mg doses of Ab6, as described in example 2. When evaluating treatment effect, inclusion of day-1 (after Ab6 infusion) in the statistical analysis indicated that a significant treatment effect occurred immediately after infusion (fig. 38). In the figure, day 0 is defined as the infusion day, and day-1 data represents the pre-infusion condition. The percentage of migraine was significantly reduced from day-1 (baseline, day prior to infusion) to day 0. Moreover, the 300mg dose had a greater magnitude of effect than the 100 mg dose, and both showed greater effect than the placebo group.

Example 7

This example relates to antibody ab10.h3 (LuAG 09222). Ab10.h3 has been studied in several clinical trials including phase I trials to determine the safety and tolerability of ascending doses, and more recently also phase II studies (termed HOPE studies, described herein before) have been conducted, showing a role in the prevention of migraine. Thus, ab10.h3 has proven to be safe and useful in treating migraine sufferers.

Ab10.H3 is described in patent application WO 2017181039, the contents of which (including the sequence listing) are incorporated herein by reference in their entirety. H3 is a humanized form of antibody Ab10 described in WO 2017181039. WO 2017181039 describes that Ab10 and ab10.h3 have increased inhibitory specificity for the PCAP1 receptor pathway relative to the VPAC1 or VPAC2 pathway, in the case of Ab10, humanization of Ab10 reduced the PACAP 38-induced PCA1-R mediated increase in cAMP IC50 (pM), whereas humanization increased PACAP 38-induced VPAC1-R mediated and VPAC 2-mediated increase in cAMP IC50 (pM), which generally indicated increased inhibitory specificity for the PCAP1 receptor. Given the abundant expression of VCAP1 and VCAP2 receptors outside the nervous system (see paragraph in WO 2017181039), reduced relative specificity for VCAP1 and VCAP2 receptors may reduce the unintended effect, i.e., interaction with undesired target cells when the antibody is used as a therapeutic or prophylactic agent, a desirable property.

Thus, humanized antibodies provide the additional technical feature of increased specificity of inhibition of the PCAP1 receptor pathway relative to the VPAC1 or VPAC2 pathway, which further complements the above-described technical differences. The following table summarizes these points from WO 2017181039. Table 2 was generated by WO 2017181039 based on the reference table from WO 2017181039.

Ab10 and Ab10.H3 antibodies provide in vivo neurological effects Ab10.H and Ab10.H3 decrease light sensitivity in PACAP-induced photophobic mouse models as shown in example 11 of WO 2017181039 (see, e.g., paragraph in WO 2017181039). Photophobia (an extreme photosensitivity) is a symptom commonly experienced by patients suffering from neurological disorders such as migraine and thus the presently claimed antibodies are antibodies that can be used to treat or prevent such neurological disorders or symptoms. In example 13 of WO 2017181039, ab10.h3 reduced trigeminal parasympathetic reflex in a rat model of vascular dysfunction of cluster headache, trigeminal neuralgia and possibly migraine, as measured by tear and nose temperature following intranasal administration of umbelliferone (see paragraph-and figures 34 and 35 in WO 2017181039). Furthermore, example 8 WO 2017181039 shows that Ab10 inhibits PACAP-induced cutaneous vasodilation in rabbits. Although the cutaneous vascular effect was studied in this experiment, the neural mechanisms of vasoconstriction/vasodilation are essentially the same in the brain, and vasodilation is one of the causative mechanisms of various types of headache. In summary, the antibodies can be used to treat or prevent neurological disorders or symptoms, such as headache.

Ab10 and Ab10.H3 recognize unique epitopes within PACAP Ab10 and Ab10.H3 recognize residues 19, 22, 23 and 27 of PACAP as shown in example 12 of WO 2017181039. Humanized forms of the presently claimed Ab10 are also expected to recognize identical residues because they share the same 6 CDR sequences with their parent Ab10. Binding to this particular epitope (particularly residues 23 and 27) appears to provide the unique and beneficial feature described therein, namely the inability to bind to PAC1 receptor-expressing cells via PACAP.

Affinity humanization of Ab10 increased the affinity for PACAP38 from 7.5E-11 to 2.9E-11 (Ab10. H), 2.2E-11 (Ab10.H2), 2.2E-11 (Ab10.H3), or 1.9E-11 (Ab10.H4) (Table 4 of WO 2017181039).

Humanization of Ab10 reduces the IC50 (pM) of PACAP 38-induced PCA1-R mediated increase in cAMP from 180.3 to 163.4 (Ab10. H), 21.3 (Ab10.H2), 30.7 (Ab10.H3), 22.8 (Ab10.H4), 22.7 (Ab10.H5) pM (see tables 2 and 3 of WO 2017181039).

Example 7

High concentration coformulation of eplerenone bezelizumab-LuAG 00922 requires a delicate balance of formulation components and pH. The correct balance of components and pH will provide a low viscosity which enables subcutaneous administration, furthermore a stable formulation with a long shelf life can be achieved. For typical monoclonal antibody (mAb) drug product formulations, conformational and colloidal stability will be optimized based on the unique biophysical properties of the specific antibody. For co-formulations of two mabs, formulation development becomes challenging when the biophysical properties of each mAb are significantly different from each other. In this case, the isoelectric point of eplerizumab is about 8.1, while the isoelectric point of LuAG00922 is about 6.9. It is often desirable to formulate mabs in compositions having a distance of a few pH units from the isoelectric point to improve colloidal stability and solubility, which is critical for stability at high concentrations. In addition, the behavior of LuAG00922 as the sole antibody component in high concentration formulations exhibited a unique pH-dependent viscosity, and at pH 5.5, when the concentration of LuAG00922 was higher than 158 mg/mL, luAG00922 formulations had a relatively high viscosity of > 70 cP. As pH increases from 5.5 to 6.0 and further to 6.5, the viscosity of luag00922 shows a change in pH-dependent charged plaque from protonated positively charged plaque at pH 5.5 to deprotonated neutral charged plaque at 6.0 and 6.5, resulting in a decrease in viscosity with increasing pH.

Experiment Design (DOE)

Formulation design space was created by varying the pH and amounts of L-histidine, sorbitol, L-arginine hydrochloride, poloxamer 188 and protein concentration. Since the desired pH range was achieved using 10 to 50mM histidine buffer, a pH range of 5.25 to 6.75 was studied, which is desirable to provide buffer capacity for mAb subcutaneous formulations. NaCl and L-arginine hydrochloride excipient levels were studied at 3 levels of 10, 80, 150 mM and are expected to be potential stabilizers, viscosity reducers and tonicity modifiers. The L-sorbitol excipient levels were chosen to be 50, 150 and 250mM and are desirable as potential stabilizers, cryoprotectants and tonicity adjusting agents. Poloxamer 188 was studied at 0.005%, 0.0275% and 0.05% (w/v) and was expected to act as a surfactant to prevent protein aggregation and particle formation. Total antibody concentration levels were studied at 100, 125 and 150 mg/mL to provide concentrations high enough to enable subcutaneous administration of the formulation. A deterministic screening experimental Design (DOE) was created using JMP statistical software, which uses 7 factors, each factor having 3 levels, yielding 18 formulations. The DOE was applied to eplerizumab-LuAG 00922 co-formulations at ratios of 1:1, 1:2, and 2:1, yielding a total of 54 formulations.

Formulation composition and buffer exchange

The reagents used to prepare the formulations are in table 3 below. A working stock solution of each formulation component was prepared by 0.20M L-histidine, 1.0M sodium chloride (NaCl), 3.5M sorbitol, 0.9M L-arginine hydrochloride, and 10.0% (w/v) poloxamer 188. The 18 formulation buffers in table 4 were produced by diluting the appropriate amount of each working stock solution for each formulation component into distilled deionized water (Milli-Q water). The final pH of each formulation buffer was adjusted using 5M hydrochloric acid, and the formulation was then filtered with a 0.22 μm PES filter.

Table 3 reagents used in the formulations.

18 Formulation buffers containing L-histidine, L-sorbitol and L-arginine hydrochloride were prepared by mixing together the appropriate volumes of working stock solutions and diluting with distilled deionized water (Milli-Q water) to obtain the compositions in the following tables. Three ratios of eplerizumab to LuAG00922 (1:2, 1:1, 2:1) were selected and each of its buffers was exchanged into 18 formulations using Big Tuna (non-chain laboratories (Unchained Labs)) to yield a total of 54 protein formulations.

Table 4-18 formulation buffer compositions at ratios of 1:2, 1:1 and 2:1 eplerenone MAb to LuAG 00922.

Stability of 54 formulations was evaluated using an ultra-high pressure liquid chromatography using size exclusion columns at the following conditions and time points.

5 ℃ C (0, 1, 4, 7 months)

25 ℃ (1, 4, 7 Months)

40 ℃ (1 Month)

Method of

Protein concentration measurement:

After buffer exchange into the desired coformulation, protein concentration was measured using Solo VPE and AGILENT CARY 60 UV-Vis spectrophotometers or Lunatic spectrophotometers (non-chain laboratories, inc.) and a280 extinction coefficients known for eplerenone and LuAG 00922.

High throughput assay:

Size exclusion chromatography and ultra high pressure liquid chromatography (SE-UPLC) were performed using a Waters acquisition H-like UPLC with TUV detector attached to a Waters BEH SEC column (P/N186005225). SE-UPLC analysis was performed at 5 ℃,25 ℃,30 ℃ and 40 ℃ at t=0 and each time point to evaluate the percentage of high molecular weight species (HMW%). Eplerenone has an initial HMW% of 0.32% and LuAG00922 has an initial HMW% of 0.94%. The total HMW% results for each of the 54 co-formulations after buffer exchange at t=0 ranged from 0.39% to 1.40%. JMP DOE model analysis showed that changes in protein concentration, eplerenone's bead mab to luAG00922 ratio, histidine concentration, L-arginine hydrochloride concentration, pH, and sorbitol concentration had a significant effect on the HMW% response. Sodium chloride concentration and poloxamer (P188) concentration did not show significant changes in HMW% response.

High-throughput viscosity measurements were performed using a bead method based on Uncle (non-chain laboratories) Dynamic Light Scattering (DLS) (He et al 2010, ANALYTICAL BIOCHEMISTRY [ analytical biochemistry ], volume 399, stage 1, pages 141-143, ,High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions [, high-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions ]). The protein formulation was incorporated into 100nm diameter polystyrene beads (thermo scientific): P/N3100A).

Table 11 eplerenone with a 1:1 ratio of Leu AG00922

。

Claims (29)

1. A pharmaceutical composition comprising eplerenone and LuAG09222, wherein eplerenone comprises a VH region as defined in SEQ ID No.: 4 and a VL as defined in SEQ ID No.: 10, and LU AG09222 comprises a VH region as defined in SEQ ID No.: 15 and a VL region as defined in SEQ ID No.: 20.

2. The pharmaceutical composition according to claim 1, formulated to maintain the bioactivity and/or storage stability of the eplerian monoclonal antibody and LuAG09222 antibody therein.

3. The pharmaceutical composition according to claim 1 or 2, which maintains the biological activity and/or storage stability of the eplerizumab and LuAG09222 antibodies therein for at least 1 month, at least 2 months, at least 3-6 months, at least 6-9 months, at least 9-12 months, or at least one year.

4. The pharmaceutical composition according to any of the preceding claims, comprising or further comprising histidine and polysorbate 80 or poloxamer 188.

5. The pharmaceutical composition according to any of the preceding claims, comprising or further comprising one, two or all of the following excipients NaCl, sorbitol and arginine.

6. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG09222 in a total concentration of 100 mg/mL to 300 mg/mL.

7. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG09222 in a total concentration of about 100 mg/mL, about 150 mg/mL, about 200 mg/mL, about 250 mg/mL or about 300 mg/mL.

8. The pharmaceutical composition of claim 6 or 7, comprising eplerenone and LuAG09222 in a ratio of about 1:1, 1:2, or 2:2.

9. The pharmaceutical composition according to any one of the preceding claims, wherein the composition comprises about 100-300 mg/mL eplerizumab and about 50-100 mg/mL LuAG09222.

10. The pharmaceutical composition according to any one of the preceding claims, wherein the composition comprises about 100 mg/mL of eplerenone and about 50 mg/mL of LuAG09222.

11. The pharmaceutical composition according to any one of the preceding claims, wherein eplerenone and LuAG0922 are the only active ingredients in the composition.

12. The pharmaceutical composition according to any of the preceding claims, wherein the concentration of histidine ranges between 10-50 mM, optionally about 20-40 mM, the concentration of poloxamer P188 ranges between 0.0025-0.0120% w/v, and the concentration of polysorbate 80 ranges between 0.005-0.05% w/v, inclusive.

13. The pharmaceutical composition according to any one of the preceding claims, wherein the concentration of NaCl is between about 10-150 mM, optionally about 30-70 mM, the concentration of sorbitol (optionally L-sorbitol) is between 50-250 mM, optionally the concentration of sorbitol is about 90-180 mM, and the concentration of arginine (optionally L-arginine) is between 50-250 mM, inclusive.

14. The pharmaceutical composition according to any of the preceding claims, wherein the pH is between about 5.0-6.8, inclusive.

15. The pharmaceutical composition according to any one of the preceding claims, wherein the pH is about 5.0, about 5.5, about 5.9, about 6.0, about 6.5 or about 6.8.

16. The pharmaceutical composition according to any of the preceding claims, which is suitable for intravenous administration or subcutaneous administration.

17. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG90222 in a ratio of about 2:1, about 1:1 or about 1:2, and comprising about 20-40 mM histidine buffer, about 90-180 mM sorbitol, 0.0025-0.0120% w/v poloxamer P188 and 30-70 mM NaCl, and having a pH of about 6, optionally about pH 5.9.

18. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG90222 in a ratio of 2:1, and further comprising about 25-35 mM histidine buffer, about 165-175 mM sorbitol, about 0.0025-0.010% w/v poloxamer P188 and about 25-35 mM NaCl, and having a pH of about 6, optionally about pH 5.9.

19. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG90222 in a ratio of 1:1, and further comprising about 30-40 mM histidine buffer, about 130-140 mM sorbitol, about 0.005-0.010% w/v poloxamer P188 and about 45-55 mM NaCl, and having a pH of about 6, optionally pH 5.9.

20. The pharmaceutical composition according to any one of the preceding claims, comprising eplerenone and LuAG90222 in a ratio of 1:2, and further comprising about 35-45 mM histidine buffer, about 90-100 mM sorbitol, about 0.010-0.0120% w/v poloxamer P188 and about 65-75 mM NaCl, and having a pH of about 6, optionally pH 5.9.

21. A pharmaceutical composition according to any one of the preceding claims for use as a medicament.

22. The pharmaceutical composition according to any one of the preceding claims for use in the treatment or prevention of headache, optionally chronic or episodic migraine or cluster headache, further optionally wherein administration of said pharmaceutical composition comprising said antibody combination has a cumulative or synergistic effect on the number, duration and/or intensity of inhibition, alleviation or prevention of migraine attacks compared to subcutaneous or intravenous administration of a pharmaceutical composition comprising the same dose of eplerizumab alone or LuAG 09222.

23. The pharmaceutical composition according to any one of the preceding claims for use in the treatment or prevention of pain, optionally any one of acute pain, chronic pain, neuropathic pain, nociceptive pain and/or neuropathic pain, further optionally wherein administration of the pharmaceutical composition comprising the antibody combination has a cumulative or synergistic effect on inhibiting or alleviating pain compared to subcutaneous or intravenous administration of a pharmaceutical composition comprising the same dose of eplerizumab alone or LuAG 09222.

24. The pharmaceutical composition according to any of the preceding claims, for administration monthly (every 4 weeks) or every 2 weeks.

25. The pharmaceutical composition according to any one of the preceding claims, wherein eplerenone is comprised of a heavy chain as defined in SEQ ID No.: 5 or SEQ ID No.: 6 and a light chain as defined in SEQ ID No.: 11.

26. The pharmaceutical composition according to any of the preceding claims, wherein LUAG09222 comprises a heavy chain as defined in SEQ ID No.: 16 or SEQ ID No.: 26 and a light chain as defined in SEQ ID No.: 21.

27. A method of treating or preventing headache, optionally chronic or episodic migraine or cluster headache, the method comprising or consisting of subcutaneously or intravenously administering a combination of eplercanizumab and LuAG09222 antibody, wherein eplercanizumab comprises a VH region as defined in SEQ ID No.: 4 and VL as defined in SEQ ID No.: 10, and LU AG09222 comprises a VH region as defined in SEQ ID No.: 15 and VL region as defined in SEQ ID No.: 20, and optionally wherein administration of said antibody combination has a cumulative or synergistic effect on the number, duration and/or intensity of inhibition, alleviation or prevention of migraine attacks compared to subcutaneous or intravenous administration of the same dose of eplercanizumab or LuAG09222 alone.

28. A method of treating or preventing pain, optionally any of acute pain, chronic pain, neuropathic pain, nociceptive pain, and/or radicular pain, comprising or consisting of subcutaneously or intravenously administering a combination of eplerian mab and LuAG09222 antibody, wherein eplerian mab comprises a VH region as defined in SEQ ID No.: 4 and a VL as defined in SEQ ID No.: 10, and LU AG09222 comprises a VH region as defined in SEQ ID No.: 15 and a VL region as defined in SEQ ID No.: 20, and optionally wherein administration of said antibody combination has an additive or synergistic effect in inhibiting or alleviating pain compared to subcutaneous or intravenous administration of the same dose of eplerian mab alone or LuAG 09222.

29. The method of claim 27 or 28, wherein the eplerenone and LuAG09222 antibody are administered subcutaneously or intravenously by administering the pharmaceutical composition of any one of claims 1-20.