Combination therapy

Abstract

The present invention relates to a composition comprising eptinezumab and LuAG09222. This composition may be used, for example, to treat or prevent headache, pain, migraine, cluster headache, or endometriosis.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 509,854, filed on 23 June 2023, the contents of which this application are incorporated in their entirety by reference.

[0002] Disclosure of sequence listings The contents of the electronic sequence listing (1143257o014013.xml, size: 25,446 bytes, creation date: June 17, 2024) are incorporated herein by reference in their entirety.

[0003] Arrays that are not allowed to be included in St.26 XML files due to their length Table A below lists the sequences that are present in the priority application, U.S. Provisional Patent Application No. 63 / 509,854 (identified above, which is incorporated herein by reference in its entirety), but which, due to their length, could not be included in the 1143257o014013.xml file submitted attached to this specification.

[0004] [Table 1]

[0005] Technical field of inventions The present invention relates to a composition comprising two antibodies or fragments thereof (including Fab fragments) having specificity for different targets. One of these antibodies or antibody fragments specifically binds to human calcitonin gene-related peptide (hereinafter referred to as "CGRP"), and the other antibody or antigen-binding fragment specifically binds to pituitary adenylyl cyclase-activating polypeptide ("PACAP"). [Background technology]

[0006] Calcitonin gene-related peptide (CGRP) is produced as a 37-amino acid long, multifunctional neuropeptide. Two forms of CGRP, CGRP-α and CGRP-β, exist in humans and possess similar activity. In humans, CGRP-α and CGRP-β differ in three amino acids and originate from different genes. CGRP is released from numerous tissues, including the trigeminal nerve, and upon activation, the neuropeptide is released within the meninges, mediating neurogenic inflammation characterized by vasodilation, vascular leakage, and mast cell degradation. (Durham, PL, New Eng. J. Med., 350(11):1073-75 (2004)). The biological action of CGRP is mediated by binding to receptor-associated membrane proteins (RAMPs) via the CGRP receptor (CGRP-R), which consists of seven transmembrane components. CGRP-R further requires the activity of receptor component proteins (RCPs), which are essential for efficient coupling to adenylyl cyclase via G proteins and for cAMP production. Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68 (2001).

[0007] Several antibodies have been approved for the treatment of migraines. These antibodies bind to either CGRP or the CGRP receptor. FDA-approved antibodies include Aimovig (erenumab), Ajovi (fremanezumab), Emgality (galcanezumab), and Vyepti (eptinezumab). This invention relates to Vyepti (eptinezumab or Ab6 as used herein), an approved agent for the prophylactic treatment of migraines. The recommended dose is 100 mg intravenously every three months. Some patients may benefit from a dose of 300 mg every three months.

[0008] Pituitary adenylyl cyclase-activating polypeptide ("PACAP") is a member of the secretin / vasoactive intestinal peptide ("VIP") / growth hormone-releasing hormone ("GHRH") family. PACAP is a multifunctional vasodilator peptide and exists in two α-amidate-active forms (containing 38 amino acids (PACAP38) and 27 amino acids (PACAP27)). Both peptides have the same N-terminal 27 amino acids and are synthesized from the same precursor protein, prepro-PACAP (see Moody et al., Curr. Opin. Endocrinol. Diabetes Obes., 18(1):61-67, 2011). PACAP38 is the more common active form and accounts for up to 90% of PACAP morphologies in mammalian tissues (see Kaiser and Russo, Neuropeptides, 47:451-461, 2013). The sequence of PACAP38 is identical in all mammals, differing from the avian and amphibian orthologues by only one amino acid (see Vaudry et al., Pharmacol. Rev., 52:269-324, 2000). The secretin / VIP / GHRH family includes the mammalian peptide histidine methionamide ("PHM"), secretin, glucagon, glucagon-like peptide-1 ("GLP1"), glucagon-like peptide-2 ("GLP2"), glucose-dependent insulinotropic polypeptide ("GIP"), and growth hormone-releasing factor ("GRF"). PACAP27 shows 68% sequence identity with VIP at the amino acid level (see Vaudry et al., 2000).

[0009] The biological effects of PACAP are mediated by three different G protein-coupled receptors: PAC1-R, vasoactive intestinal peptide receptor 1 ("VPAC1-R"), and vasoactive intestinal peptide receptor 2 ("VPAC2-R"). These receptors are expressed in a variety of tissues. PAC1-R is particularly abundant in the nervous system (e.g., olfactory bulb, thalamus, hypothalamus, cerebellum, and spinal cord dorsal horn), pituitary gland, and adrenal gland. In contrast, VPAC1-R and VPAC2-R are mainly expressed in the lungs, liver, and testes, but have also been detected in other tissues. VPAC1-R expression has been detected in the nervous system (e.g., cerebral cortex and hippocampus), lungs, liver, intestinal smooth muscle cells, megakaryocytes, and platelets. VPAC1-R binds to receptor-associated membrane proteins ("RAMP" proteins, particularly RAMP2) (see Christopoulos et al., J. Biol. Chem., 278:3293-3297, 2002). VPAC2-R expression profiles include the nervous system (e.g., thalamus, hippocampus, brainstem, and dorsal root ganglia (DRGs)), cardiovascular system, digestive system, pancreas, and reproductive system (see Usdin et al., Endocrin., 135:2662-2680, 1994; Sheward et al., Neurosci., 67:409-418, 1995).

[0010] PACAP is thought to be involved in a variety of conditions and disorders, including, but not limited to, migraines, headaches, and pain. Migraines are thought to have a neurovascular component. Migraines affect about 10% of the adult population in the United States and are typically accompanied by severe headaches. About 20-30% of migraine sufferers experience aura, including focal neurological phenomena, that precede and / or accompany the onset of their migraine. Several observations suggest that PACAP plays a role in migraines: (1) In humans, plasma PACAP concentrations are elevated during migraine attacks (attack phase) compared to the interictal period (see Tuka et al., Cephalalgia, 33(13):1085-1095, 2013), (2) PACAP38 infusion induced headaches in healthy subjects and migraine-like attacks in migraine patients (see Schytz et al., Brain, 132:16-25, 2009 and Amin et al., Brain, 137:779-794, 2014), (3) PACAP-induced vasodilation may be involved in neurogenic inflammation (Kaiser and See Russo, Neuropeptides, 47:451-461, 2013), and (4) PACAP-induced migraines are accompanied by photosensitivity, phonosensitivity, and nausea, and respond to triptans (see Amin et al., Brain, 32:140-149, 2012). PACAP has also been shown to induce vasodilation, photosensitivity, mast cell degranulation, and neuronal activation (see Markovics et al., Neurobiology of Disease, 45:633-644, 2012; Baun et al., Cephalalgia, 32(4):337-345, 2012; Chan et al., Pharmacology & Therapeutics, 129:332-351, 2011).

[0011] Furthermore, PACAP may be involved in diseases and disorders other than migraines, headaches, and pain. For example, PACAP may be correlated with or play a causal role in anxiety disorders (International Publication No. 2012 / 106407), thrombocytopenia (International Publication No. 2004 / 062684), and inflammatory skin diseases (International Publication No. 2010 / 007175). PACAP and PAC1-R polymorphisms have been associated with post-traumatic stress disorder ("PTSD"), major depressive disorder, and generalized anxiety disorder in women, suggesting a role of PACAP in these conditions. Furthermore, supporting the role of PACAP in thrombocytopenia, there is an excess of PACAP in patients with trisomy 18, and they exhibit impaired megakaryocyte maturation (see Schytz et al., 2010; Moody et al., Curr. Opin. Endocrinol. Diabetes Obes., 18(1):61-67, 2011).

[0012] PACAP and other neuropeptides such as calcitonin gene-related peptide (CGRP), substance P, neurokinin A, bradykinin, and endothelin-1 are also expressed in the lower urinary tract (LUT) (see Arms and Vizzard, Handbook Exp. Pharmacol., 202:395-423, 2011), and have been reported to be potentially involved in urinary tract disorders such as LUT dysfunction and urinary tract infections ("UTIs"), voiding abnormalities, urinary urgency, nocturia, urinary incontinence, overactive bladder, and pain associated with these conditions.

[0013] PACAP and PACAP receptors have been suggested to regulate inflammatory and neuropathic pain and are involved in both pain facilitation and pain inhibition (see Davis-Taber et al., J. Pain, 9(5):449-56, 2008). Furthermore, PACAP has been reported to be required for spinal cord desensitization and the induction of neuropathic pain (see Mabuchi et al., J. Neurosci., 24(33):7283-91, 2004). In addition, morphine withdrawal behavior has been reported to be altered in PACAP receptor-deficient mice, suggesting the involvement of PACAP in the anxiolytic response during morphine withdrawal (see Martin et al., Mol. Brain Res., 110(1):109-18, 2003).

[0014] The present invention relates to an anti-PACAP antibody called LuAG09222, Ab10.H3 or ALD1910 (all synonyms of the same antibody) (Moldovan Loomis et al., J Pharmacol Exp Ther 369:26-36, April 2019). This agent has been tested at different doses in various clinical trials regarding migraine and has just shown remarkable results in a proof-of-concept trial for migraine (clinicaltrials.gov number NCT05133323 and The Pharma Letter 20 Apr 2023 “Lundbeck posts positive Phase IIa results with LuAG09222”).

[0015] The present invention relates to the combined use of eptinezumab and LuAG09222 as a combination therapy or the preferred use as a composition containing both agents. Said combination therapy or composition can be used for the treatment of medical conditions, optionally headache, episodic or chronic migraine, cluster headache, endometriosis or pain. SUMMARY OF THE INVENTION

[0016] This disclosure provides a method for treating headaches in patients, which comprises administering to patients in need an effective amount of an anti-CGRP antibody (e.g., eptinezumab) or a fragment thereof, and an effective amount of an anti-PACAP antibody (e.g., LuAG09222) or a fragment thereof, as disclosed herein.

[0017] According to one embodiment, the present invention relates to the treatment of migraines. The antibody treatment may be initiated during the interictal period, i.e., during a migraine attack, or during the attack period, i.e., during a migraine episode. The migraine may include, for example, chronic migraine or episodic migraine, and in a particular aspect of the present invention, the patient suffers from chronic migraine. In the present invention, the anti-CGRP antibody is denoted as Ab6, eptinezumab, or Vyepti, and these all refer to the same antibody. The anti-PACAP antibody is denoted as LuAG09222, ALD1910, or Ab10.H3, and these all refer to the same antibody.

[0018] The antibodies of the present invention may be produced in yeast or mammalian cells, such as Pichia pastoris or CHO cells. The antibodies of the present invention may be in a composition with a pH of 5.0 to 6.8, comprising histidine (10-50 mM), polysorbate 80, or poloxamer 188 (0.005-0.05% w / v). This composition may further comprise an osmotic regulator, optionally NaCl (10-150 mM), sorbitol (50-250 mM), and / or arginine (50-250 mM). The total amount of both antibodies may be 150 mg / ml, for example, 100 mg / ml of eptinezumab and 50 mg / ml of LuAG09222. The formulation may be administered to the patient subcutaneously or intravenously (iv). Further embodiments of the formulation and co-formulations are described herein and in the claims. In the present invention, histidine includes L-type histidine (L-histidine), sorbitol includes L-sorbitol type, and arginine includes L-arginine type.

[0019] In another embodiment of the present invention, the combination therapy or composition is useful in a manner for the relief, treatment or prevention of migraines (with or without aura), cancer or tumors, angiogenesis associated with the growth of cancer or tumors, angiogenesis associated with the survival of cancer or tumors, weight loss, pain, hemiplegic migraine, cluster headache, migraine-like neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic paroxysmal hemiplegia, secondary headaches resulting from underlying structural problems of the head and neck, occipital neuralgia, sinus headache (e.g., associated with sinusitis), and allergy-induced headache or migraine. The antibodies and antibody fragments of the present invention are particularly useful in treating, preventing, improving, controlling or reducing the risk of one or more of the following conditions or diseases: overactive bladder and bladder infections, other urinary tract disorders including pain, chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, eye pain, toothache, postoperative pain, trauma-related pain, burn-related pain, diabetes mellitus, non-insulin-dependent diabetes mellitus and other inflammatory autoimmune diseases, vascular disorders, inflammation, arthritis, bronchial hypersensitivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, traumatic brain injury, epilepsy, neurodegenerative diseases, skin diseases (such as pruritus, neurogenic skin redness, rosacea and erythema), inflammatory bowel disease, irritable bowel syndrome, cystitis, dysmenorrhea, and other diseases that can be treated, prevented, or alleviated by antagonizing CGRP and / or PACAP signaling. Of particular importance are the acute or prophylactic treatment of headaches, including migraines and cluster headaches, other pain-related disorders, and endometriosis.

[0020] In another embodiment of the present invention, the combination therapy or composition is useful in a manner aimed at reducing, treating, or preventing gastroesophageal reflux and / or visceral pain associated with gastroesophageal reflux, indigestion, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis, or pancreatitis.

[0021] In migraines, hyperactivation of the trigeminal nerve increases the release of calcitonin gene-related peptide (CGRP) and other peptides, which in turn triggers the release of neurogenic inflammatory mediators. These mediators further increase CGRP synthesis and release over time, corresponding to a typical migraine episode. CGRP mediates its effects by vasodilation of cerebral arteries and arterioles, primarily activating adenylyl cyclase in smooth muscle cells in the trigeminal vascular network, which leads to abnormal activation of nociceptors and the sensation of pain.

[0022] Pituitary adenylyl cyclase-activated polypeptide (PACAP) is thought to be involved in the pathophysiology of headaches based on several lines of evidence. Studies have demonstrated that PACAP levels are elevated during migraine attacks compared to inter-attack periods in migraine patients. Furthermore, PACAP is thought to be released during migraine attacks, and PACAP levels are elevated in patients with chronic migraines.

[0023] Evidence from several other lines has also been obtained from provocation studies, in which both PACAP-38 and PACAP-27 induced migraine-like events in both healthy subjects and migraine patients. PACAP may also be involved in cluster headaches. Similarly, studies have demonstrated that PACAP levels are elevated during cluster events compared to interictal periods.

[0024] Although CGRP and PACAP neuropeptides overlap in location and exhibit physiological similarities, differences in their biological effects are observed.

[0025] This suggests that dual neutralization of CGRP and PACAP peptides yields a significant clinical effect compared to single-target neutralization in patients suffering from migraines / headaches.

[0026] Both eptinezumab and LuAG09222 are humanized antibodies genetically engineered against human CGRP and PACAP, respectively. By binding to these neuropeptides, eptinezumab and LuAG09222 can inhibit their binding to their respective receptors, thereby inhibiting downstream signaling that can cause headache and pain sensations.

[0027] These antibodies are IgG1κ immunoglobulins containing a human constant region. The variable regions of the light and heavy chains consist of human sequences and humanized rabbit sequences.

[0028] Eptinezumab is a prescription medication used to treat migraines in adults. It is administered as a 30-minute intravenous (IV) infusion at doses of 100 or 300 mg. Patients receive this medication four times a year (i.e., every three months).

[0029] LuAG09222 binds to human PACAP with high affinity. LuAG09222 inhibits migraine-related effects (photosensitivity, vasodilation, increased body temperature, and lacrimation) and can reduce pain in preclinical mechanical models. Data from clinical trials have confirmed the safety, tolerability, and pharmacokinetics of LuAG09222. In a human challenge trial, LuAG09222 demonstrated that it prevents PACAP38-induced vasodilation, as well as PACAP38-induced facial flushing and increased heart rate. A Phase 2 trial of LuAG09222 for migraines (HOPE trial) provided evidence of the effectiveness of PACAP targeting in migraine prevention. Data from the Phase 2 HOPE trial (NCT05133323) revealed the potential efficacy of LuAG09222 as a migraine preventive agent. Overall, the above trials met their primary endpoints, and significant differences were observed between the high-dose treatment group over a 12-week double-blind period. This multinational, multicenter trial included 237 individuals with episodes or chronic migraines that had not responded to 2-4 previous prophylactic medications. Participants were randomly assigned to receive either 750 mg (n=97), 100 mg of LuAG09222 (n=46), or placebo (n=94) for 4 weeks, followed by 12 weeks of safety follow-up. At the end of the 4-week period, the investigators observed a 2.0-day difference in the reduction of monthly migraine days (MMD) between the high-dose treatment group and the placebo group (95% CI, -3.5 to -0.6, P=0.0106).

[0030] Compositions comprising both products developed for subcutaneous administration (eptinezumab and LuAG09222) provide additive therapeutic benefits in headache and non-headache pain disorders, offering a differentiated and / or superior treatment option. Clear segments / phenotypes in migraine (patients with inadequate response to anti-CGRP agents, patients with parasympathetic symptoms, patients with high-frequency / chronic migraines, and alternative / adjunct agents to anti-CGRP agents) and episodic cluster headache include subjects with elevated circulating CGRP and PACAP levels.

[0031] In yet another embodiment of the present invention, the combination therapy or composition is useful for the treatment, improvement or prevention of chronic or recurrent migraines, cluster headaches, endometriosis, or pain.

[0032] In certain embodiments, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222, wherein eptinezumab has a VH region described in SEQ ID NO: 4 and a VL region described in SEQ ID NO: 10, and LuAG09222 has a VH region described in SEQ ID NO: 15 and a VL region described in SEQ ID NO: 20.

[0033] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, which is formulated to maintain the biological activity and / or storage stability of the eptinezumab and LuAG09222 antibody contained herein.

[0034] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the eptinezumab and LuAG09222 antibody contained herein maintain the biological activity and / or storage stability for at least one month, at least two months, at least three months, at least three to six months, at least six to nine months, at least nine to twelve months, or at least one year.

[0035] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, further comprising histidine and either polysorbate 80 or poloxamer 188.

[0036] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, comprising one, two or all of the following excipients: NaCl, sorbitol, and arginine, or further comprising the pharmaceutical composition comprising the above.

[0037] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the total concentration of eptinezumab and LuAG09222 is between 100 mg / mL and 300 mg / mL.

[0038] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the total concentration of eptinezumab and LuAG09222 is 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or 300 mg / mL.

[0039] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, having an eptinezumab to LuAG09222 ratio of 1:1, 1:2, or 2:2.

[0040] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the pharmaceutical composition comprises 100 to 300 mg / mL of eptinezumab and 50 to 100 mg / mL of LuAG09222.

[0041] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the pharmaceutical composition comprises 100 mg / mL of eptinezumab and 50 mg / mL of LuAG09222.

[0042] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein eptinezumab and LuAG0922 are the sole active ingredients in the composition.

[0043] The pharmaceutical composition according to any one of the preceding claims, wherein histidine is in a concentration of 10 to 50 mM, optionally in the range of 20 to 40 mM, poloxamer P188 is in the range of 0.0025 to 0.0120% w / v, and polysorbate 80 is in the range of 0.005 to 0.05% w / v (including both extreme values).

[0044] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the concentration of NaCl is 10 to 150 mM, optionally 30 to 70 mM, the concentration of sorbitol (e.g., L-sorbitol) is 50 to 250 mM, optionally 90 to 180 mM, and the concentration of arginine (e.g., L-arginine) is in the range of 50 to 250 mM.

[0045] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the pH is 5.0 to 6.8 (including both extreme values).

[0046] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the pH is 5.0, 5.5, 5.9, 6.0, 6.5, or 6.8.

[0047] A pharmaceutical composition according to any one of the preceding claims, which is suitable for intravenous or subcutaneous administration.

[0048] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the eptinezumab and LuAG09222 are present in a ratio of 2:1, 1:1, or 1:2, and the composition comprises 20-40 mM histidine buffer, 90-180 mM sorbitol, 0.0025-0.0120% w / v poloxamer P188, and 30-70 mM NaCl, and has a pH of about 6, optionally pH 5.9.

[0049] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the eptinezumab and LuAG09222 are present in a 2:1 ratio, and the composition comprises 25-35 mM histidine buffer, 165-175 mM sorbitol, 0.0025-0.010% w / v poloxamer P188, and 25-35 mM NaCl, and has a pH of about 6, optionally pH 5.9.

[0050] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the eptinezumab and LuAG09222 are present in a 1:1 ratio, and the composition comprises 30-40 mM histidine buffer, 130-140 mM sorbitol, 0.005-0.010% w / v poloxamer P188, and 45-55 mM NaCl, and has a pH of about 6, optionally pH 5.9.

[0051] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, wherein the eptinezumab and LuAG09222 are present in a 1:2 ratio, and the composition comprises 35-45 mM histidine buffer, 90-100 mM sorbitol, 0.010-0.0120% w / v poloxamer P188, and 65-75 mM NaCl, and has a pH of about 6, optionally pH 5.9.

[0052] In another specific embodiment, the present invention provides a pharmaceutical composition for use as a pharmaceutical, comprising eptinezumab and LuAG09222 as described above.

[0053] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222, for use in the treatment or prevention of headache, optionally chronic or episodic migraine or cluster headache, as described above.

[0054] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, for use in the treatment or prevention of pain.

[0055] In another specific embodiment, the present invention provides a pharmaceutical composition comprising eptinezumab and LuAG09222 as described above, for administration monthly (every four weeks) or every two weeks.

[0056] In another specific embodiment, the present invention provides a pharmaceutical composition or use thereof comprising eptinezumab and LuAG09222 as described above, wherein eptinezumab has a heavy chain as described in SEQ ID NO: 5 or SEQ ID NO: 6 and a light chain as described in SEQ ID NO: 11.

[0057] In another specific embodiment, the present invention provides a pharmaceutical composition or use thereof comprising eptinezumab and LuAG09222 as described above, wherein LUAG09222 has a heavy chain as described in SEQ ID NO: 16 or SEQ ID NO: 26 and a light chain as described in SEQ ID NO: 21.

[0058] In another specific embodiment, the present invention provides a method for treating or preventing headache, optionally chronic or episodic migraine or cluster headache, the method comprising subcutaneous or intravenous administration of a combination of eptinezumab and the LuAG09222 antibody, wherein eptinezumab has a VH region as described in SEQ ID NO: 4 and a VL region as described in SEQ ID NO: 10, and LuAG09222 has a VH region as described in SEQ ID NO: 15 and a VL region as described in SEQ ID NO: 20, and optionally, administration of the antibody combination has an additive or synergistic effect on inhibiting, reducing or preventing the frequency, duration and / or intensity of migraine episodes compared to subcutaneous or intravenous administration of eptinezumab or LuAG09222 alone.

[0059] In another specific embodiment, the present invention provides a treatment or prevention of pain, the method comprising subcutaneous or intravenous administration of a combination of eptinezumab and the LuAG09222 antibody, wherein eptinezumab has a VH region as described in SEQ ID NO: 4 and a VL region as described in SEQ ID NO: 10, and LuAG09222 has a VH region as described in SEQ ID NO: 15 and a VL region as described in SEQ ID NO: 20, and optionally, administration of the antibody combination has an additive or synergistic effect on inhibiting or reducing pain compared to subcutaneous or intravenous administration of eptinezumab or LuAG09222 alone.

[0060] In another specific embodiment, the present invention provides a method for treating or preventing headache, optionally chronic or episodic migraine or cluster headache, by administering eptinezumab and LuAG09222, wherein eptinezumab and the LuAG09222 antibody are administered subcutaneously or intravenously using the pharmaceutical composition described in the prior embodiments.

[0061] In another specific embodiment, the present invention provides a method for treating or preventing pain, such as acute pain, chronic pain, neuropathic pain, nociceptive pain, and / or radiculopathy, by administering eptinezumab and LuAG09222, wherein eptinezumab and the LuAG09222 antibody are administered subcutaneously or intravenously using the pharmaceutical composition described in the prior embodiments. [Brief explanation of the drawing]

[0062] [Figure 1] The number of subjects in the human clinical trials described in Example 1 who were treated with either Ab6 (treatment group) or placebo and showed a 50%, 75%, or 100% reduction in migraine at each monitoring point throughout the period is shown. The graph on the right for each group corresponds to patients who received 1000 mg of Ab6, and the graph on the left for each group corresponds to the corresponding placebo control. In each response rate group, patients who received Ab6 had a significantly higher response rate than the placebo-treated control, with p-values ​​of 0.0155, 0.0034, and 0.0006 in each group, as shown. [Figure 2]The graph shows the median (±QR)% change from baseline in the number of migraine days per month in the placebo and Ab6 treatment groups over 12 weeks after treatment (p=0.0078). The upper and lower lines represent the results for patients treated with placebo and 1000 mg Ab6, respectively. [Figure 3] The graphs show the median (±QR)% change from baseline in the number of migraine episodes per month in the placebo and Ab6 treatment groups over 12 weeks after treatment. The upper and lower lines represent the results for patients treated with placebo and 1000 mg Ab6, respectively. [Figure 4] The graphs show the median (±QR)% change from baseline in the monthly migraine duration in the placebo and Ab6 treatment groups over 12 weeks after treatment. The upper and lower lines represent the results for patients treated with placebo and 1000 mg Ab6, respectively. [Figure 5] Summarize patient screening, allocation to treatment and control groups, and patient loss throughout the follow-up period. [Figure 6] We will compare HIT-6 responder analyses of the Ab6 treatment group and the placebo group at baseline, 4 weeks post-treatment, 8 weeks post-treatment, and 12 weeks post-treatment. [Figure 7] This shows the proportion of patients who showed only “some” or “little / no” headache effects at baseline and after Ab6 administration in the HIT-6 analysis. At baseline, most patients were “substantial” or “severe” affected by migraines. At each subsequent time point, a significantly larger proportion of patients who received 1000 mg Ab6 had only “some” or “little / no” HIT-6 effects compared to the placebo control (right bar in each group, red). [Figure 8] This includes the pharmacokinetic (PK) profile of Ab6 administered intravenously at a single dose of 1000 mg. [Figure 9]The plasma-free pharmacokinetic (PK) parameters for a single 1000 mg intravenous dose of Ab6 are shown in the table. N (number of patients), mean, and standard deviation (SD). The parameters and units are Cmax (μg / mL), AUC0-∞ (mg*hr / mL), half-life (days), Vz (L), and CL (mL / hr). [Figure 10] The change from baseline (mean ± SEM) in the number of migraine days per month as a single dose for Ab6 (1000 mg iv) versus placebo in the study described in Example 1 is shown. [Figure 11] The time-averaged number of migraine days (+ / SD) for the entire analysis population for the trial described in Example 1 is shown. The 21-27 day e-diaries were completed by applying normalization to the visit interval and multiplying the observed frequency by the reciprocal of the completion rate. [Figure 12] The distribution of the actual number of migraine days and changes in the Ab6 treatment group during weeks 1 to 4 for the trial described in Example 1 is shown below. [Figure 13] The distribution of the actual number of migraine days and changes in the placebo group during weeks 1 to 4 for the trial described in Example 1 is shown below. [Figure 14] The distribution of the actual number of migraine days and changes in the Ab6 treatment group during the 5-8 week period for the trial described in Example 1 is shown below. [Figure 15] The distribution of the actual number of migraine days and changes in the placebo group during the 5-8 week period for the trial described in Example 1 is shown below. [Figure 16] The distribution of the actual number of migraine days and changes in the Ab6 treatment group during the 9-12 week period for the trial described in Example 1 is shown below. [Figure 17] The distribution of the actual number of migraine days and changes in the placebo group during weeks 9-12 for the trial described in Example 1 is shown below. [Figure 18]The 50% responder rates for Ab6 and the placebo treatment group in the trial described in Example 1 are shown. Subjects whose migraine frequency decreased by 50% or more were considered 50% responders. The 21-27 day e-diary was completed by applying normalization to the visit interval and multiplying the observed frequency by the reciprocal of the completion rate. [Figure 19] The 75% responder rates for Ab6 and the placebo treatment group in the trial described in Example 1 are shown. Subjects whose migraine frequency decreased by 75% or more were considered 75% responders. Normalization was applied as described in Figure 18. [Figure 20] The 100% responder rates for Ab6 and the placebo treatment group in the trial described in Example 1 are shown. Subjects with a 100% reduction in migraine frequency are considered 100% responders. Normalization was applied as described in Figure 18. [Figure 21] This shows the time-averaged migraine severity for the entire analysis population in the study described in Example 1. On the scale used, a mean migraine score of 3 represents "moderate headache." [Figure 22] This section summarizes the changes from baseline in the measured attributes of the placebo and treatment groups in the trial described in Example 1. [Figure 23] The graph shows the percentage of patients with migraines on days 1, 7, 14, 21, and 28 in the clinical trial described in Example 2, in the 300 mg, 100 mg, and placebo treatment groups. The top line represents the placebo result, the bottom line represents the 300 mg dose result, and the middle line represents the 100 mg dose result. [Figure 24] The percentage of patients in the 300 mg and 100 mg treatment groups who achieved a 50% reduction in migraine days at 1 month, 1-3 months (after the first infusion), and 4-5 months (after the second infusion) in the clinical trial described in Example 2 is shown. In each graph, the data bars show the results for the 100 mg, 300 mg, and placebo groups from left to right. Statistical significance is as shown. ++ indicates a statistically significant difference compared to placebo, + indicates a statistically significant difference compared to placebo (unadjusted), and § indicates a statistically significant difference compared to placebo (post-hoc). [Figure 25] The percentage of patients in the 300 mg and 100 mg treatment groups who achieved a 75% reduction in migraine days at 1 month, 1–3 months (after the first infusion), and 4–5 months (after the second infusion) in the clinical trial described in Example 2 is shown. The order of the data and the statistical significance labels are as shown in Figure 24. [Figure 26] The percentage of patients in the 300 mg and 100 mg treatment groups who achieved a 100% reduction in migraine days at 1 month, 1–3 months (after the first infusion), and 4–5 months (after the second infusion) in the clinical trial described in Example 2 is shown. The order of the data and the statistical significance labels are as shown in Figure 24. [Figure 27] The characteristics of patients in each treatment group in the clinical trial described in Example 3 are summarized below. *Following the American Academy of Neurology / American Headache Society guidelines for migraine prevention and treatment (drugs identified by clinical review of coded medical data); SD, standard deviation; BMI, body mass index. [Figure 28] The graph shows the difference from placebo in the mean number of migraine days (MMD) from baseline over 1–3 months in different baseline subgroups of human clinical trials for patients with chronic migraines. In the graph, data points represent the mean, and the lines show the 95% confidence interval (CI) of the change from placebo for each subgroup treated with 100 mg (upper line) or 300 mg (lower line), as shown on the left. [Figure 29] Differences from placebo in the change from baseline in mean migraine days (MMD) over 1–3 months in different baseline subgroups of human clinical trials for patients with sudden-onset migraines. [Figure 30] Change from baseline in mean migraine days (MMD) over two dose intervals in chronic migraine patients with at least one day of acute medication use per month at baseline. Triangle: Placebo (n=366). Circle: 100 mg Ab6 / dose (n=356). Square: 300 mg Ab6 / dose (n=350). [Figure 31]Mean number of days of acute medication use per month in chronic migraine patients who had at least one day of acute medication use per month at baseline. Triangle: Placebo (n=366). Circle: 100 mg Ab6 / dose (n=356). Square: 300 mg Ab6 / dose (n=350). [Figure 32] Change from baseline in acute drug use by subgroups of chronic migraine patients with different baseline days of acute drug use. Solid line: Patients with 10 or more days of acute drug use per month at baseline. Dashed line: Patients with at least 1 day and less than 10 days of acute drug use per month at baseline. Triangle: Placebo. Circle: 100 mg Ab6 / dose. Square: 300 mg Ab6 / dose. [Figure 33] Summary of acute drug days by subgroup in chronic migraine patients with baseline acute drug use. [Figure 34] Change from baseline in mean migraine days (MMD) over two dose intervals in patients with sudden migraine who had at least one day of acute medication use per month at baseline. Triangle: Placebo (n=222). Circle: 100 mg Ab6 / dose (n=221). Square: 300 mg Ab6 / dose (n=222). [Figure 35] Mean number of days of acute medication use per month in patients with sudden migraine who had at least one day of acute medication use per month at baseline. Triangle: Placebo (n=222). Circle: 100 mg Ab6 / dose (n=221). Square: 300 mg Ab6 / dose (n=222). [Figure 36] Change from baseline in acute drug use by subgroups of chronic migraine patients with different baseline durations of spontaneous drug use. Solid line: Patients with 10 or more days of acute drug use per month at baseline. Dashed line: Patients with at least 1 day and less than 10 days of acute drug use per month at baseline. Triangle: Placebo. Circle: 100 mg Ab6 / dose. Square: 300 mg Ab6 / dose. [Figure 37] Summary of acute drug days by subgroup in patients with sudden migraine and baseline acute drug use. [Figure 38]The migraine data includes day -1. Day 0 is defined as the injection day. Therefore, the data for day 0 shows the therapeutic effect after the injection. [Figure 39] Based on clinical data from a Phase II trial using LuAG09222 (Ab10.H3), specifically in patients with migraines, dosage prediction was performed using R software (nlmixr). The drug was administered monthly for 7 months, after which it was discontinued. [Modes for carrying out the invention]

[0063] Detailed explanation By combining population PK models of eptinezumab and LuAG09222, we were able to validate a PKPD model to determine the ratio of eptinezumab to LuAG09222 required to suppress the release of each target (CGRP and PACAP) to less than 50%. Dosages were explored by simultaneously administering both compounds as bolus doses. The administration frequency was set to once a month, and the route of administration was subcutaneous.

[0064] The terminal elimination half-life of eptinezumab is 27 days, and it is used at 100 or 300 mg for the treatment of migraines, while the terminal elimination half-life of LuAG09222 is approximately 15 days.

[0065] To achieve less than 50% of the free target with monthly sc administration intervals, it is estimated that 100 mg of eptinezumab and 50 mg of LuAG09222 will be required.

[0066] No treatment-related deaths or adverse events attributable to the pharmacological activity of eptinezumab or LuAG09222 were observed in monotherapy, single-dose or repeated-dose studies in rats or cynomolgus monkeys. No effects on reproductive function or behavior, fertility, or early embryonic development were observed in rats after administration of eptinezumab. No evidence of parental effects, fetal death, fetal toxicity, or teratogenicity was found in rats or rabbits with administration of eptinezumab or LuAG09222. No effects on F1 generation survival, physical development, behavior, or reproductive behavior were observed in prenatal and postnatal developmental studies of eptinezumab in rats.

[0067] Throughout all non-clinical studies, the NOEL / NOAEL for eptinezumab or LuAG09222 administered intravenously as monotherapy was the highest dose administered (up to 150 mg / kg / dose).

[0068] Since both eptinezumab and LuAG09222 are humanized monoclonal antibodies, pharmacokinetic interactions from the administration of combination formulations or concurrent administration are not expected.

[0069] definition It should be understood that the present invention is not limited to specific methodologies, protocols, cell lines, animal species or genera and reagents described (as they may vary). It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the appended claims. The singular forms “a,” “an,” and “it” used herein include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to “cell” includes multiple such cells, and a reference to “protein” includes one or more proteins and equivalents known to those skilled in the art. All technical and scientific terms used herein have the same meaning as they would be commonly understood by those skilled in the art to which the present invention belongs, unless it is clearly indicated otherwise.

[0070] As used herein, the term “chronic migraine” refers to a condition in which a patient experiences headaches on average at least 15 days per month, with some of these headaches meeting the ICHD-3 criteria for migraine (with or without aura). The term “episodic migraine” refers to a condition in which a patient experiences headaches on average less than 15 days per month, with typically 4 to 15 of these headaches being of the ICHD-3 definition of migraine (with or without aura).

[0071] As used herein, the term “diagnosed with chronic migraine” refers to a patient who meets the clinical criteria for chronic migraine, regardless of whether a formal diagnosis has been made for that patient. The term “diagnosed with episodic migraine” refers to a patient who meets the clinical criteria for episodic migraine, regardless of whether a formal diagnosis has been made for that patient.

[0072] As used herein, the term “intravenous administration” refers to a method of administration in which a substance, such as an antibody, is directly introduced into the patient’s circulation, most typically into venous circulation. The substance may be introduced in a carrier fluid, optionally an aqueous solution, such as ordinary saline. The substance may be administered in a single formulation or multiple formulations, provided that the administration is completed within a short time (e.g., within 1 day, preferably within 12 hours, more preferably within 6 hours, most preferably within 1-2 hours). The term “subcutaneous administration” (or sc administration) refers to a method of administration in which a substance, such as an antibody, is administered into the dermis, a layer of skin just below the epidermis and dermis. Subcutaneous administration can be performed in various locations, such as the outer part of the upper arm and abdomen, the front of the thigh, the upper back, or the upper buttocks behind the hip bone.

[0073] As used herein, the term “baseline migraine days” refers to the number of days a patient experiences migraines during a specified period (e.g., before treatment). For example, baseline migraine days can be determined by recording, for instance, whether or not a migraine occurred each day over a period of one month or longer.

[0074] As used herein, the term “migraine days per month” means the number of days per month on which a patient has a migraine, i.e., the number of days on which, at any point in time, the patient has symptoms that meet the clinical definition of a migraine. The migraine days per month can be determined by recording each day whether or not a migraine occurred.

[0075] As used herein, the term “monthly headache days” means the number of days per month on which a patient has a headache attack, i.e., the number of days on which, at any point in the day, the patient has symptoms that meet the clinical definition of a headache. Monthly headache days can be determined by recording whether or not a headache occurred each day.

[0076] Calcitonin gene-related peptide (CGRP): As used herein, CGRP encompasses the following Homo sapiens CGRP-α and Homo sapiens CGRP-β amino acid sequences available from American Peptides (Sunnyvale, CA) and Bachem (Torrance, CA).

[0077] CGRP-α:ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH2 (SEQ ID NO: 22), where the terminal phenylalanine is amidated.

[0078] CGRP-β:ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH2 (SEQ ID NO: 23), where the terminal phenylalanine is amidated, is also any membrane-bound form of these CGRP amino acid sequences, as well as mutants, splice variants, isoforms, orthologues, homologs, and variants of this sequence.

[0079] Pituitary adenylate cyclase-activated polypeptide (PACAP): As used herein, unless otherwise specified, PACAP includes all mammalian types of PACAP, and in particular includes the amino acid sequences of Homo sapiens PACAP27 and Homo sapiens PACAP38.

[0080] PACAP38: HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 24), and any variants, splice variants, isoforms, orthologues, homologs, and variants of this sequence.

[0081] PACAP27: HSDGIFTDSYSRYRKQMAVKKYLAAVL (Sequence ID 25), and any variants, splice variants, isoforms, orthologues, homologs, and variants of this sequence.

[0082] Expression Vectors: These DNA vectors contain elements that facilitate the manipulation of foreign protein expression in target host cells, e.g., yeast or mammalian cells, or optionally Pichia pastoris or CHO cells. Conveniently, the manipulation of sequences for transformation and DNA production are first performed in a bacterial host, e.g., Escherichia coli, and typically the vector will contain sequences to facilitate such manipulation, including a bacterial origin of replication and appropriate bacterial selection markers. The selection markers encode proteins necessary for the survival or growth of transformed host cells grown in the selection 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) confer resistance to antibiotics or other toxins, (b) compensate for nutritional deficiencies, or (c) supply essential nutrients not available from the complex medium. Exemplary vectors and methods for yeast transformation are described, for example, in Burke, D., Dawson, D., & Stearns, T. (2000). Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Plainview, NY: Cold Spring Harbor Laboratory Press.

[0083] Expression vectors for use in yeast or mammalian cells will generally further include yeast or mammalian-specific sequences containing selectable nutritional requirements or drug markers for identifying transformed yeast strains or transformed mammalian cells. Drug markers may further be used to amplify the copy number of the vector in host cells.

[0084] The target polypeptide coding sequence is operably ligated to a transcriptional and translational regulatory sequence that provides polypeptide expression in a host cell, e.g., Pichia pastoris or CHO cells. These vector components may include, but are not limited to, one or more of the following: enhancer elements, promoters, and transcription termination sequences. Sequences for polypeptide secretion, e.g., signal sequences, may also be included. Since the expression vector is often integrated into the host cell genome, the yeast or mammalian origin of replication is optional. In one embodiment of the present invention, the target polypeptide is operably ligated or fused to a sequence that provides optimized secretion of the polypeptide from yeast diploid cells.

[0085] Nucleic acids are "operably linked" when they are placed in a functional relationship with another nucleic acid sequence. For example, the DNA of a signal sequence is operably linked to the DNA of a polypeptide when it is expressed as a preprotein involved in polypeptide secretion, and a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences to be linked are adjacent, and in the case of a secretion leader, adjacent and within the reading frame. However, enhancers do not need to be adjacent. This is achieved by linking at a convenient restriction site or via PCR / recombination methods well known to those skilled in the art (Gateway® Technology; Invitrogen, Carlsbad California). If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0086] Promoters are untranslated sequences located upstream (5') of the start codon of a structural gene (generally within about 100–1000 bp) that control the transcription and translation of a specific nucleic acid sequence to which they are operably linked. Such promoters are classified into several classes: inductive, constitutive, and repressive promoters (which increase transcription levels in response to the absence of repressors). Inductive promoters can initiate increased levels of transcription from DNA under their control in response to several changes in culture conditions, such as the presence or absence of nutrients or changes in temperature.

[0087] Promoter fragments can also function as sites for homologous recombination and integration of expression vectors into the same location in the host genome, or a selectable marker can be used as the site for homologous recombination. Examples of suitable promoters from Pichia include the AOX1 promoter (Cregg et al. (1989) Mol.Cell.Biol.9:1316-1323), the ICL1 promoter (Menendez et al. (2003) Yeast 20(13):1097-108), the glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al. (1997) Gene 186(1):37-44), and the FLD1 promoter (Shen et al. (1998) Gene 216(1):93-102). The GAP promoter is a strong constitutive promoter, while the AOX and FLD1 promoters are inducible.

[0088] Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, Pyk, and chimeric promoters derived therefrom. In addition, non-yeast promoters, and optionally mammalian, insect, plant, reptile, amphibian, viral, and avian promoters, may be used in the present invention. Most typically, the promoters will include mammalian promoters (which may be endogenous with respect to the expressed gene) or yeast or viral promoters that provide efficient transcription in the yeast system.

[0089] Examples of mammalian promoters include, among others, promoters derived from cytomegalovirus (CMV), chicken 3-actin (CBM), adenomatous polyposis coli (APC), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) promoter, CAG promoter, β-actin promoter, elongation factor-1 (EF1) promoter, early growth response 1 (EGR-1) promoter, eukaryotic initiation factor 4A (EIF4A1) promoter, monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, and human gene promoters. Optionally, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters may be used. Two or more combinations of the aforementioned promoters may be used. Furthermore, inducible promoters may be used. The use of inductive promoters provides a molecular switch that can turn on the expression of an operatively linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0090] The antibodies of the present invention can also be produced as fusion polypeptides with heterologous polypeptides (e.g., signal sequences having a specific cleavage site at the N-terminus of a mature protein or polypeptide, or other polypeptides). Generally, the signal sequence may be a component of a vector or part of a polypeptide coding sequence inserted into a vector. The selected heterologous signal sequence is preferably recognized and processed through one of the standard pathways available in the host cell. The budding yeast (S. cerevisiae) α-factor prepro-signal has been proven effective for the secretion of various recombinant proteins from P. pastoris. Other yeast signal sequences include α-conjugation factor signal sequences, invertase signal sequences, and signal sequences derived from other secretory yeast polypeptides. In addition, these signal peptide sequences may be engineered to provide enhanced secretion in diploid yeast expression systems. Secretory signals used in mammalian and yeast cells include mammalian signal sequences that may be heterologous to the secreted protein or may be native sequences of the secreted protein. The signal sequence includes a prepeptide sequence, and in some examples, may include a propeptide sequence. Many signal sequences found on immunoglobulin chains are known in the art, including, for example, the K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chains, and human Ig light chains. See, for example, Hashimoto et al. Protein Eng 11(2)75(1998) and Kobayashi et al. Therapeutic Apheresis 2(4)257(1998).

[0091] Transcription can be increased by inserting transcription activator sequences into a vector. These activators are typically cis-acting elements of DNA, approximately 10–300 bp in length, that act on the promoter to increase its transcription. Transcription enhancers are relatively orientation and position-independent and are found at the 5' and 3' ends of the transcription unit, within introns, and within the coding sequence itself. Enhancers can be spliced ​​into the expression vector at the 5' and 3' ends of the coding sequence, but are preferably located 5' from the promoter.

[0092] Expression vectors used in eukaryotic host cells may contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available in the untranslated regions of eukaryotic or viral DNA or cDNA, specifically from the 3' end of the translation termination codon. These regions contain nucleotide fragments that are transcribed as polyadenylated fragments within the untranslated portion of mRNA.

[0093] The construction of a suitable vector containing one or more of the components listed above is performed using standard ligation techniques or PCR / recombination methods. The isolated plasmid or DNA fragment is cleaved, prepared, and rejoined in the desired form to produce the required plasmid, or is produced via a recombination method. For analysis to confirm the correct sequence in the constructed plasmid, host cells are transformed using the ligation mixture, and, where appropriate, transformants that have successfully resisted antibiotics (e.g., ampicillin or zeosin) are selected. Plasmids derived from the transformants are prepared and analyzed and / or sequenced by restriction endonuclease digestion.

[0094] As an alternative to fragment restriction and ligation, DNA sequences can be inserted into vectors using recombination methods based on att sites and recombinant enzymes. Such methods are known to those skilled in the art, for example, as described by Landy (1989) Ann. Rev. Biochem. 58:913-949. Such methods utilize intermolecular DNA recombination mediated by a mixture of recombinant proteins encoded by lambda and Escherichia coli (E. coli). Recombination occurs between specific binding (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, Weisberg, ed. (Cold Spring Harbor, NY: Cold Spring Harbor Press), pp. 211-250. DNA segments adjacent to the recombination site are exchanged, so that after recombination, the att site is a hybrid sequence consisting of sequences donated by each parent vector. Recombination can occur between DNAs of any topology.

[0095] The Att site can be introduced into a target sequence by methods such as ligating the target sequence into an appropriate vector, generating a PCR product containing the attB site using specific primers, or generating a cDNA library cloned into an appropriate vector containing the att site.

[0096] As used herein, folding refers to the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. Appropriate folding is typically the arrangement of a polypeptide that yields optimal biological activity, and in the case of antibodies, can be conveniently monitored by assays for activity (e.g., antigen binding).

[0097] The expression host can be further modified by introducing sequences encoding one or more enzymes that enhance folding and disulfide bond formation, such as foldases, chaperonins, etc. Such sequences can be constitutively or inductively expressed in yeast host cells using vectors, markers, etc., known in the art. Preferably, sequences containing sufficient transcriptional regulatory elements for the desired expression pattern are stably incorporated into the yeast genome by targeted methodologies.

[0098] For example, eukaryotic PDI is not only an efficient catalyst for protein cysteine ​​oxidation and disulfide bond isomerization, but also exhibits chaperone activity. Co-expression of PDI can promote the production of active proteins containing multiple disulfide bonds. Expression of BIP (immunoglobulin heavy chain binding protein), cyclophyllin, and others is also of interest. In one embodiment of the present invention, each of the singular parental strains expresses a different folding enzyme; for example, one strain may express BIP, while other strains may express PDI or a combination thereof.

[0099] The terms “desired protein” or “desired antibody” are used interchangeably and generally refer to a target-specific parental antibody, i.e., CGRP, PACAP, or chimeric or humanized antibody as described herein, or its binding portion derived therefrom. The term “antibody” is intended to include any polypeptide chain-containing molecular structure having a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. Prototype antibody molecules are immunoglobulins, and IgG in particular from all sources, e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, other mammals, chickens, other birds, etc., are considered “antibodies.” Numerous antibody coding sequences are described, and others can be prepared 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., scFvs), camel antibodies, nanobodies, IgNARs (shark-derived single-chain antibodies), small module immunotherapy drugs (SMIPs), and antibody fragments such as Fabs, Fab', and F(ab')2.See Streltsov VA, et al., Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype, Protein Sci. 2005 Nov;14(11):2901-9. Epub 2005 Sep 30; Greenberg AS, et al., A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks, Nature. 1995 Mar 9;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, Mol Immunol. 2001 Aug;38(4):313-26; Hamers-Casterman C, et al., Naturally occurring antibodies devoid of light chains, Nature. 1993 Jun 3;363(6428):446-8; Gill DS, et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol. 2006 Dec;17(6):653-8. Epub 2006 Oct 19.

[0100] For example, antibodies or antigen-binding fragments can be produced by genetic engineering. In this technique, as with other methods, antibody-producing cells are sensitized to the desired antigen or immunogen. Messenger RNA isolated from the antibody-producing cells is used as a template to produce cDNA using PCR amplification. A library of vectors, each containing one heavy-chain gene and one light-chain gene that retains the initial antigen specificity, is prepared by inserting appropriate sections of the amplified immunoglobulin cDNA into an expression vector. A combinatorial library is constructed by combining the heavy-chain gene library with the light-chain gene library. This results in a library of clones co-expressing the heavy and light chains (similar to Fab fragments or antigen-binding fragments of antibody molecules). Vectors containing these genes are co-transfected into host cells. If antibody gene synthesis is induced in the transfected host, the heavy and light-chain proteins self-assemble to produce an active antibody that can be detected by screening with the antigen or immunogen.

[0101] The target antibody coding sequences include those encoded by the natural sequence, as well as nucleic acids and their variants whose sequences are not identical to those disclosed due to the degeneracy of the gene code. Variant polypeptides may include amino acid (aa) substitutions, additions, or deletions. Amino acid substitutions may be conservative substitutions to eliminate non-essential amino acids, for example, by modifying glycosylation sites, or by minimizing misfolding due to the substitution or deletion of one or more cysteine ​​residues not required for function. Variants may be designed to retain or have enhanced biological activity in specific regions of the protein (e.g., functional domains, catalytic amino acid residues). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Polypeptides modified using conventional molecular biological techniques to improve their resistance to proteolysis, optimize their solubility, or make them more suitable as therapeutic agents are also included in the present invention.

[0102] Chimeric antibodies are obtained from antibody-producing cells of one species, and their variable light chain and heavy chain regions (V L and V H ) can be produced by recombinant methods by combining constant light chain and heavy chain regions from another species. Typically, chimeric antibodies utilize a rodent or rabbit variable region and a human constant region to produce an antibody that primarily has a human domain. The production of such chimeric antibodies is well known in the art and can be achieved by standard means (for example, as described in U.S. Patent No. 5,624,659, which is incorporated herein by reference in its entirety). Furthermore, it is intended that the human constant region of the chimeric antibody of the present invention may be selected from the IgG1, IgG2, IgG3, and IgG4 constant regions.

[0103] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, incorporating only the complementarity-determining regions of animal-derived antibodies. This is achieved by carefully examining the sequence of the hypervariable loop in the variable region of the monoclonal antibody and adapting it to the structure of the human antibody chain. While seemingly complex, this process is actually straightforward. See, for example, U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0104] In addition to whole immunoglobulins (or their recombinant counterparts), immunoglobulin fragments containing epitope binding sites (e.g., Fab', F(ab')2, or other fragments) can be synthesized. “Fragments” or minimal immunoglobulins can be designed using recombinant immunoglobulin technology. For example, “Fv” immunoglobulins for use in the present invention can be produced by synthesizing fused variable light chain regions and variable heavy chain regions. Combinations of antibodies, such as diabodies containing two different Fv specificities, are also of interest. In another embodiment of the present invention, SMIPs (small molecule immunopharmaceuticals), camel antibodies, nanobodies, and IgNARs are encompassed by immunoglobulin fragments.

[0105] Immunoglobulins and their fragments can be post-translation modified to add, for example, effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent substances, radioactive substances, or chemiluminescent moieties, or specific binding moieties such as streptavidin, avidin, or biotin can be used in the methods and compositions of the present invention. Examples of additional effector molecules are provided below.

[0106] A polynucleotide sequence "corresponds" to a polypeptide sequence (i.e., a polynucleotide sequence "codes" a polypeptide sequence) if the translation of the polynucleotide sequence according to the gene code results in a polypeptide sequence, and if two sequences code for the same polypeptide sequence, one polynucleotide sequence "corresponds" to another polynucleotide sequence.

[0107] A “heterogeneous” region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in relation to the larger, naturally occurring molecule. Therefore, if a heterogeneous region codes for a mammalian gene, the gene will typically be adjacent to DNA that is not adjacent to the mammalian genomic DNA in the genome of the source organism. Another example of a heterogeneous region is a construct whose coding sequence itself is not found naturally (e.g., a cDNA containing introns or a synthetic sequence with codons different from the naturally occurring gene). Allele mutations or naturally occurring mutation events do not result in heterogeneous regions of DNA as defined herein.

[0108] A "coding sequence" is the in-frame sequence of codons corresponding to a protein or peptide sequence (from a genetic coding perspective). Two coding sequences correspond to each other if the sequence or its complementary sequence codes for the same amino acid sequence. A coding sequence associated with an appropriate regulatory sequence can be transcribed and translated into a polypeptide. Polyadenylation signals and transcription termination sequences will typically be located on the 3' side of the coding sequence. A "promoter sequence" is a DNA regulatory region that can bind to RNA polymerase in the cell and initiate the transcription of the downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for the binding of regulatory molecules (e.g., transcription factors) that affect the transcription of the coding sequence. When RNA polymerase binds to the promoter sequence in the cell and transcribes the coding sequence into mRNA, the coding sequence is "under the control" of the promoter sequence or "operationally linked" to the promoter, and the mRNA is then translated into the protein encoded by the coding sequence.

[0109] Vectors are used to introduce foreign substances, such as DNA, RNA, or proteins, into organisms or host cells. Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for polypeptides). A "DNA vector" is a replicon such as plasmids, phages, or cosmids, to which another polynucleotide segment can be attached, resulting in replication of the attached segment. An "expression vector" is a DNA vector containing regulatory sequences that direct polypeptide synthesis by a suitable host cell. This typically means that a promoter binds to RNA polymerase and initiates mRNA transcription, and a ribosome binding site and initiation signal direct the translation of mRNA into polypeptides. Incorporation of a polynucleotide sequence into an expression vector at the appropriate site and with the correct reading frame, followed by transformation of a suitable host cell by the vector, enables the production of polypeptides encoded by the polynucleotide sequence.

[0110] The "amplification" of a polynucleotide sequence is the in vitro production of multiple copies of a specific nucleic acid sequence. The amplified sequence is usually in the form of DNA. A variety of techniques for performing such amplification are described in a review article by Van Brunt (1990, Bio / Technol., 8(4):291-294). Polymerase chain reaction, or PCR, is the prototype of nucleic acid amplification, and the use of PCR herein should be considered an example of other appropriate amplification techniques.

[0111] The general structure of antibodies in vertebrates is now well understood (Edelman, GM, Ann. NYAcad. Sci., 190:5 (1971)). An antibody consists of two identical polypeptide light chains ("light chains") with a molecular weight of approximately 23,000 daltons and two identical heavy chains ("heavy chains") with a molecular weight of 53,000 to 70,000. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chains enclosing the heavy chains that begin at the mouth of the "Y" configuration. The "branching" portion of the "Y" configuration is F ab The region is called the stem part of the "Y" three-dimensional arrangement, F C This is called a 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 chain. The N-terminus has a variable region that is specific to the antigen that induced it, and is approximately 100 amino acids long, with slight variations between the light and heavy chains and from antibody to antibody.

[0112] The variable region in each chain is linked to a constant region that extends the rest of the chain length, and the variable region of a particular class of antibody does not change depending on the specificity of the antibody (i.e., the antigen that induces it). There are five known major classes of constant regions that determine the class of an immunoglobulin molecule (γ, μ, α, δ, and ε (gamma, mu, alpha, delta, or epsilon) corresponding to the heavy chain constant regions of IgG, IgM, IgA, IgD, and IgE). The constant region or class determines the subsequent effector function of the antibody, including complement activation (Kabat, EA, Structural Concepts in Immunology and Immunochemistry, 2nd Ed., pp. 413-436, Holt, Rinehart, Winston (1976)) and other cellular responses (Andrews, DW, et al., Clinical Immunobiology, pp. 1-18, WBSanders (1980); Kohl, S., et al., Immunology, 48:187 (1983)), while the variable region determines the antigen it reacts to. Light chains are classified as either kappa (κ) or lambda (λ). Each heavy chain class can be prepared with either a kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and when immunoglobulins are produced by either hybridomas or B cells, the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds.

[0113] The term "variable region" or "VR" refers to the domains within each pair of light and heavy chains of an antibody that are directly involved in the binding of the antibody to the antigen. Each heavy chain has a variable domain (V) at one end. H It has a variable domain (V) at one end. L The light chain has a constant domain at the other end, the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.

[0114] The terms “complementarity-determining region,” “hypervariable region,” or “CDR” refer 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, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include hypervariable regions as defined by Kabat et al. (“Sequences of Proteins of Immunological Interest,” Kabat E., et al., US Dept. of Health and Human Services, 1983) or hypervariable loops in the three-dimensional structure of the antibody (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). The CDRs of each chain are held in close proximity by framework regions and, together with CDRs from other chains, contribute to the formation of antigen-binding sites. Within the CDR, there are select amino acids described as selectivity-determining regions (SDRs) that represent important contact residues used by the CDR in antibody-antigen interactions (Kashmiri, S., Methods, 36:25-34 (2005)). In this invention, when a specific antibody amino acid or nucleic acid residue is referred to by number, this generally refers to its position within the specific amino acid or nucleic acid sequence (i.e., a specific sequence identifier) ​​and / or its position according to Kabat et al.'s numbering.

[0115] The terms "framework region" or "FR" refer to one or more framework regions within the variable region of the antibody's light or heavy chain (see Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These terms include amino acid sequence regions interposed between CDRs within the variable regions of the antibody's light and heavy chains.

[0116] 「Cmax」refers to the maximum (or peak) concentration achieved by an antibody or other compound in a test area (e.g., another compartment such as serum or cerebrospinal fluid) after the drug has been administered. For example, serum Cmax can be measured from serum, which is prepared, for example, by collecting a blood sample, coagulating it, and separating the solid components by centrifugation or other means to produce serum (blood without blood cells or clotting factors), and then detecting the concentration of the analyte in the serum by ELISA or other means known in the art.

[0117] 「AUC」refers to the area under the concentration-time curve, expressed in units of mg / mL×hr (or equivalently mg×hr / ml) unless otherwise specified.「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.

[0118] 「I max 」refers to the maximum pharmacodynamic response induced by an anti-CGRP antibody dose, preferably 350 mg or more, more typically at least 750 or 1000 mg, compared to the response induced by a lower anti-CGRP antibody dose. For example, such a response can be detected by inhibition of vasodilation after topical application of capsaicin.

[0119] Sequence of antibody Ab6 (eptinezumab) Antibody Ab6 (eptinezumab) contains the variable light chain sequence described below. QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR (SEQ ID NO: 10)

[0120] Antibody Ab6 (eptinezumab) contains the light chain sequence described below.

Chemical Structure

[0121] The antibody Ab6 (eptinezumab) contains the variable heavy chain described below. EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS(Sequence ID 4)

[0122] The antibody Ab6 (eptinezumab) contains the heavy chain sequence described below. [ka]

[0123] Alternatively, the heavy chain of Ab6 (eptinezumab) may lack C-terminal lysine, meaning the heavy chain sequence may include the sequence described below. [ka]

[0124] In one embodiment of the present invention described herein (hereinafter), the Fab fragment may be produced by enzymatic digestion of Ab6 (e.g., papain). In another embodiment of the present invention, Ab6 or its Fab fragment may be produced by expression in mammalian cells, e.g., CHO, NSO, or HEK 293 cells, fungi, insects, or microbial systems, e.g., yeast cells (e.g., diploid yeast, e.g., diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0125] The CDR region of Ab6 (eptinezumab) is outlined below.

[0126] Light chain CDR1 QASQSVYHNTYLA (Sequence ID 7)

[0127] Light chain CDR2 DASTLAS (Sequence ID 8)

[0128] Light chain CDR3 LGSYDCTNGDCFV (Sequence ID 9)

[0129] Heavy-chain CDR1 GYYMN (Sequence ID 1)

[0130] Heavy-chain CDR2 VIGINGATYYASWAKG (Sequence ID 2)

[0131] Heavy-chain CDR3 GDI (SEQ ID NO: 3)

[0132] Antibody LuAG09222 LuAG09222 contains the following variable heavy chain sequence. EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSS (Sequence ID 15)

[0133] LuAG09222 contains the heavy chain sequence described below. [ka]

[0134] According to some aspects of the present invention, LuAG009222 may have terminal lysine in the heavy chain as described below. [ka]

[0135] LuAG09222 contains a variable light chain sequence that includes the sequence described below. DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAWFQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKR(Sequence ID 20)

[0136] LuAG09222 contains a light chain sequence that includes the sequence described below. [ka]

[0137] In one embodiment of the present invention described herein (described later), the Fab fragment may be produced by enzymatic digestion of Ab10.H3 (e.g., papain). In another embodiment of the present invention, LuAG09222 may be produced by expression in mammalian cells, e.g., CHO, NSO, or HEK 293 cells, fungi, insects, or microbial systems, e.g., yeast cells (e.g., diploid yeast, e.g., diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0138] The CDR area of ​​LuAG09222 is outlined below.

[0139] Light chain CDR1 QSSESVYGNYLA (Sequence ID 17)

[0140] Light chain CDR2 EASKLES (Sequence ID 18)

[0141] Light chain CDR3 AGGDISEGVA (Sequence ID 19)

[0142] Heavy-chain CDR1 SYYMT (SEQ ID NO: 12)

[0143] Heavy-chain CDR2 FIDAGGDAYYASWAKG (SEQ ID NO: 13)

[0144] Heavy-chain CDR3 DLDL (SEQ ID NO: 14)

[0145] As described herein, antibodies and fragments thereof may be post-translationally modified to include 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 streptavidin, avidin, biotin, cytotoxins, cytotoxic materials, and radioactive materials.

[0146] Antibodies or fragments thereof may also be chemically modified to provide further advantages such as increased polypeptide solubility, stability, and circulation time (in vivo half-life) or decreased immunogenicity (see U.S. Patent No. 4,179,337). Chemical moieties for derivatization may be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, and polyvinyl alcohol. Antibodies and fragments thereof may be modified at random or predetermined positions within the molecule and may contain one, two, three or more conjugated chemical moieties.

[0147] The polymer can have any molecular weight and can be branched or unbranched. In the case of polyethylene glycol, a preferred molecular weight is about 1 kD to about 100 kDa for ease of handling and manufacture (the term "about" indicates that in preparations of polyethylene glycol, some molecules are heavier and some are lighter than the stated molecular weight). Other sizes may be used depending on the desired therapeutic profile (e.g., desired duration of sustained release, effect on biological activity (if any), ease of handling, degree or absence of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). For example, polyethylene glycol is available in approximately 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, and 15,000. It may have an average molecular weight of 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. Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the respective disclosures of which are incorporated herein by reference.

[0148] Numerous bonding methods are available to those skilled in the art. For example, see European Patent No. 0401384, incorporated herein by reference (binding of PEG to G-CSF). See also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently bonded to amino acid residues via reactive groups such as free amino or carboxyl groups. Reactive groups are those to which activated polyethylene glycol molecules can be bonded. Amino acid residues having free amino groups may include lysine residues and N-terminal amino acid residues, and amino acid residues having free carboxyl groups may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Sulfhydryl groups can also be used as reactive groups for bonding polyethylene glycol molecules. Bonding at amino groups, e.g., N-terminal or lysine groups, is preferred for therapeutic purposes.

[0149] As suggested above, polyethylene glycol can be bound to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to polypeptides via covalent bonding to lysine, histidine, aspartic acid, glutamic acid, or cysteine ​​residues. One or more reaction chemistry methods are used to link polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or two or more types of amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof).

[0150] Alternatively, the antibody or fragment thereof may have an increased in vivo half-life through fusion with albumin (including, but not limited to, recombinant human serum albumin or its fragments or variants (see, for example, U.S. Patent No. 5,876,969 issued March 2, 1999, European Patent No. 0413622, and U.S. Patent No. 5,766,883 issued June 16, 1998, whose wholes are incorporated herein by reference)) or other circulating blood proteins, such as transferrin or ferritin. In preferred embodiments, the polypeptide and / or antibody (including its fragments or variants) of the present invention is fused with 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 No. 0322094, whose wholes are incorporated herein by reference). Polynucleotides encoding the fusion proteins of the present invention are also encompassed by the present invention.

[0151] Regarding detectable parts, further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, β-galactosidase, and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansilchloride. Further exemplary chemiluminescent parts include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include iodine-125( 125 I), carbon-14 ( 14 C), sulfur 35( 35 S), tritium ( 3 H) and phosphorus 32 ( 32 P) is one example, but it is not limited to these.

[0152] Regarding the functional components, exemplary cytotoxic agents include methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, alkylating agents such as mechloretamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechloretamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine. Platinum(II) (DDP) cisplatin and carboplatin (paraplatin), anthracyclines include daunorubicin (formerly daunomicin), doxorubicin (adriamycin), detrubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisanthren, antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mitramycin and anthramycin (AMC), and mitotic inhibitors include, but are not limited to, vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (Taxol), lysine, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicine, dihydroxyanthracinedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

[0153] Further 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 derived from plants and bacteria, such as ricin, diphtheria toxin, and Pseudomonas toxin, can be conjugated to humanized or chimeric antibodies or their conjugated fragments to produce cell-type specific toxic agents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[0154] Other cytotoxic agents include cytotoxic ribonucleases, such as those described by Goldenberg in U.S. Patent No. 6,653,104. Embodiments of the present invention also relate to radioimmune complexes in which a radionuclide emitting alpha or beta particles is stably bound to an antibody or its binding fragment, with or without the use of a complex-forming agent. Such radionuclides include β-emitters, for example, 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 α-emitter, for example, astatine-211( 211 At), lead-212( 212 Pb), Bismuth-212212 Bi) or -213( 213 Bi) or Actinium-225 225 It includes Ac).

[0155] For example, methods for conjugating an antibody or its binding fragment to a detectable portion are known in the art, such as those described by Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al, J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982).

[0156] The embodiments described herein further include variants and equivalents substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camel antibodies, nanobodies, IgNARs, polypeptides, variable regions, and CDRs described herein. These may include, for example, conservative substitution mutations (i.e., substitution of one or more amino acids with similar amino acids). For example, a conservative substitution refers to the substitution of another amino acid within the same general class, such as 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 neutral amino acid. The intended effects of conservative amino acid substitutions are well known in the art.

[0157] In another embodiment, the present invention intends a polypeptide sequence having at least 90% sequence homology to one or more polypeptide sequences of antibody fragments, variable regions, and CDRs described herein. More preferably, the present invention intends a polypeptide sequence having at least 95% sequence homology, more preferably at least 98% sequence homology, and more preferably at least 99% sequence homology to one or more polypeptide sequences of antibody fragments, variable regions, and CDRs described herein. Methods for determining homology between nucleic acid sequences and amino acid sequences are well known to those skilled in the art.

[0158] Another embodiment of the present invention intends these polynucleotides incorporated into an expression vector for expression in mammalian cells, e.g., CHO, NSO, HEK-293, or fungal, insect, or microbial systems, e.g., yeast cells, e.g., Pichia yeast. Suitable Pichia species include, but are not limited to, Pichia pastoris. In one embodiment of the present invention described herein (hereinafter), the Fab fragment may be produced by enzymatic digestion of Ab6 (e.g., papain) after expression of the full-length polynucleotide in a suitable host. In another embodiment of the present invention, an anti-CGRP antibody, e.g., Ab6 or its Fab fragment may be produced via expression of the Ab6 polynucleotide in mammalian cells, e.g., CHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems, e.g., yeast cells (e.g., diploid yeast, e.g., diploid Pichia) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0159] Host cells and vectors containing the aforementioned polynucleotides are also intended.

[0160] The present invention further intends to provide a vector comprising a polynucleotide encoding variable heavy and light chain polypeptide sequences and individual complementarity-determining regions (CDRs or hypervariable regions) as described herein, and a host cell comprising the said vector sequence. In one embodiment of the present invention, the host cell is a yeast cell. In another embodiment of the present invention, the yeast host cell belongs to the genus Pichia.

[0161] Method for producing antibodies and their fragments In another embodiment, the present invention intends a method for 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 No. 2009 / 0022659 to Olson et al. and U.S. Patent No. 7,935,340 to Garcia-Martinez et al. (each disclosure is incorporated herein by whole reference). Methods for producing antibodies and fragments thereof in mammalian cells, such as CHO cells, are more well known in the Art.

[0162] Other methods for producing antibodies are also well known to those skilled in the art. For example, methods for producing chimeric antibodies are now well known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.; Morrison et al., PNASUSA, 81:8651-55 (1984); Neuberger, MS et al., Nature, 314:268-270 (1985); Boulianne, G. Let al., Nature, 312:643-46 (1984) (each disclosure is incorporated herein by reference in whole)).

[0163] Similarly, other methods for producing humanized antibodies are now well known in the art (e.g., U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 to Queen et al; U.S. Patent Nos. 5,225,539 and 6,548,640 to Winter; U.S. Patent Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al; U.S. Patent No. 6,632,927 to Adair; Jones, PT et al, Nature, 321:522-525 (1986); Reichmann, L., et al, Nature, 332:323-327 (1988); Verhoeyen, M, et al. See al, Science, 239:1534-36 (1988) (each disclosure is incorporated herein by reference in its entirety).

[0164] Administration "Pharmaceutical composition" refers to a chemical or biological composition suitable for administration to mammals. Such compositions may be formulated specifically for administration by one or more of several routes, including intravenous or subcutaneous administration. Administration may be given every two weeks, once a month (every four weeks), every two months, or every three months.

[0165] A “pharmaceutical excipient” or “pharmaceutically acceptable excipient” is a carrier, usually a liquid, on which an active therapeutic agent is formulated. In one embodiment of the present invention, the active therapeutic agent is a humanized antibody or one or more fragments thereof as described herein. Excipients generally do not provide any pharmacological activity to the formulation but may provide chemical and / or biological stability and release properties. An exemplary formulation is, for example, Remington's Pharmaceutical Sciences, 19 th It can be found in Ed., Grennaro, A., Ed., 1995 (incorporated by reference).

[0166] As used herein, “pharmaceutically acceptable carriers” or “excipients” include any and all physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders. 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 immediate preparation of sterile injection solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is intended. Co-active compounds may also be incorporated into the compositions.

[0167] Pharmaceutical compositions typically need to be sterile and stable under manufacturing and storage conditions. The present invention intends for the pharmaceutical composition to exist in a lyophilized form. This composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. The present invention further intends for the pharmaceutical composition to contain a stabilizer. Appropriate fluidity can be maintained, for example, in the case of a dispersion, by maintaining the required particle size and by using a surfactant.

[0168] In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Extended absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition. Furthermore, alkaline polypeptides can be formulated into time-release formulations, for example, in compositions containing sustained-release polymers. Active compounds can be prepared using carriers that protect the compound from rapid release, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid, polyglycolic acid copolymer (PLG) can be used. Many methods for preparing such formulations are known to those skilled in the art.

[0169] According to the present invention, LuAG09222 and eptinezumab can be co-formulated according to the following embodiments.

[0170] In the following embodiments, unless otherwise specified, the total amount of eptinezumab and LuAG09222 in the pharmaceutical formulation is in the range of 100 mg / mL to 300 mg / mL (optionally about 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or 300 mg / mL), and the ratio of eptinezumab to LuAG09222 may vary. In one embodiment, the ratio of eptinezumab to LuAG09222 is 1:1, 1:2, and 2:1. For example, a 1:1 ratio in a 100 mg / mL formulation means that it contains 50 mg / mL of eptinezumab and 50 mg / mL of LuAG09222. Embodiments of the pharmaceutical composition according to the present invention are shown below.

[0171] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab 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, 0.0025-0.0120% w / v poloxamer P188, and 30-70 mM NaCl, and having a pH of about 6 (optionally pH 5.5-6.4, optionally pH 5.9).

[0172] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, 25-35 mM histidine buffer, 165-175 mM sorbitol, 0.0025-0.010% w / v poloxamer P188, and 25-35 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0173] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, and comprising 30 mM histidine buffer, 169 mM sorbitol, 0.005% w / v poloxamer P188, and 30 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of eptinezumab and LuAG90222 in the composition may be 113 mg / mL.

[0174] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, and 30-50 mM histidine buffer, 80-150 mM sorbitol, 150-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eptinezumab and LuAG90222 in the composition may be 140-150 mg / mL.

[0175] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, 30 mM histidine buffer, 80 mM sorbitol, 150 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eptinezumab and LuAG90222 may be about 150 mg / mL.

[0176] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, and 50 mM histidine buffer, 150 mM sorbitol, 250 mM L-arginine hydrochloride, and 0.02% poloxamer P188, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eptinezumab and LuAG90222 may be about 145 mg / mL.

[0177] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:1 ratio, 30-40 mM histidine buffer, 130-140 mM sorbitol, 0.005-0.010% w / v poloxamer P188, and 45-55 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0178] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:1 ratio, and containing 34 mM histidine buffer, 135 mM sorbitol, 0.008% w / v poloxamer P188, and 48 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of eptinezumab and LuAG90222 may be 120 mg / mL.

[0179] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:1 ratio, and comprising 30 mM histidine buffer, 80 mM sorbitol, 150 mM L-arginine, and 0.02% w / v poloxamer P188, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eptinezumab and LuAG90222 may be about 130 mg / mL.

[0180] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:1 ratio, and 10 mM histidine buffer, 10 mM sorbitol, 50 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of eptinezumab and LuAG90222 may be about 140 mg / mL.

[0181] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:2 ratio, 35-45 mM histidine buffer, 90-100 mM sorbitol, 0.010-0.0120% w / v poloxamer P188, and 65-75 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0182] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 1:2 ratio, and comprising 38 mM histidine buffer, 96 mM sorbitol, 0.0114% w / v poloxamer P188, and 69 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of eptinezumab and LuAG90222 may be 129 mg / mL.

[0183] In the embodiments described above, arginine (optionally L-arginine) may be added at concentrations of 10 to 150 mM, or optionally 10 mM, 50 mM, 80 mM, 100 mM, 120 mM, or 150 mM.

[0184] Furthermore, in embodiments containing arginine, the pharmaceutical formulation may contain eptinezumab and LuAG90222 in a ratio selected from 1:2, 1:1, or 2:1, and further contain 30-50 mM histidine buffer, 50-250 mM sorbitol, 10-150 mM NaCl, 0.005-0.05% w / v poloxamer 188, and 10-150 mM L-arginine hydrochloride, and have a pH of approximately 6 (optionally pH 5.9).

[0185] In another embodiment, the pharmaceutical formulation may contain eptinezumab and LuAG90222 in a ratio selected from 1:2, 1:1, or 2:1, and also contain 30-50 mM histidine buffer, 150-250 mM sorbitol, 80-150 mM NaCl, 0.005-0.05% w / v poloxamer P188, and 80-150 mM L-arginine hydrochloride, and have a pH of approximately 6 (optionally pH 5.9). The total protein concentration of eptinezumab and LuAG90222 may be 125-150 mg / mL.

[0186] In another embodiment, the pharmaceutical formulation may contain eptinezumab and LuAG90222 in a 1:2 ratio, and also contain 10-50 mM histidine buffer, 50-250 mM sorbitol, 10-150 mM NaCl, 0.005-0.05% w / v poloxamer P188, and 10-150 mM L-arginine hydrochloride, and have a pH of approximately 6 (optionally pH 5.9). The total protein concentration of eptinezumab and LuAG90222 may be 100-150 mg / mL.

[0187] In another embodiment, the pharmaceutical formulation may contain eptinezumab and LuAG90222 in a ratio selected from 1:2, 2:1, and 1:1, and also contain 10-50 mM histidine buffer, 50-250 mM sorbitol, 10-150 mM NaCl, 0.005-0.05% w / v poloxamer P188, and 10-150 mM L-arginine hydrochloride, and have a pH of about 7 (optionally pH 6.75) or about 5 (optionally pH 5.25). The total protein concentration of eptinezumab and LuAG90222 may be in the range of 100-125 mg / mL.

[0188] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab 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, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eptinezumab and LuAG90222 may be 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0189] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab 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, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eptinezumab and LuAG90222 may be 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0190] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab 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, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of eptinezumab and LuAG90222 may be 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0191] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab 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, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eptinezumab and LuAG90222 may be 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL, or 160 mg / mL).

[0192] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a ratio selected from 1:1 or 1:2, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eptinezumab and LuAG90222 may be 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL, or 160 mg / mL).

[0193] According to one embodiment, the present invention relates to a pharmaceutical composition comprising eptinezumab and LuAG90222 in a 2:1 ratio, and 30-50 mM histidine buffer, 80-150 mM sorbitol, 50-250 mM L-arginine hydrochloride, and 0.02% w / v poloxamer P188, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of eptinezumab and LuAG90222 may be 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0194] Further exemplary embodiments Embodiment 1 (E1) A composition comprising eptinezumab and LuAG09222.

[0195] E2. The composition according to E1, further comprising histidine and either poloxamer 188 or polysorbate 80.

[0196] E3. The compositions according to E1 and E2, further comprising one, two or all of the following excipients: NaCl, sorbitol (e.g., L-sorbitol), and arginine (e.g., L-arginine).

[0197] E4. A pharmaceutical composition according to any of the above embodiments, wherein the total concentration of eptinezumab and LuAG09222 is 100 mg / mL to 300 mg / mL.

[0198] E5. The pharmaceutical composition according to any of the above embodiments, wherein the total concentration of eptinezumab and LuAG09222 is 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or 300 mg / mL.

[0199] The pharmaceutical composition according to Embodiment 4 or 5, having an eptinezumab to LuAG09222 ratio of E6.1:1, 1:2, or 2:1.

[0200] A pharmaceutical composition according to any of the above embodiments, comprising E7. 100-300 mg / mL of eptinezumab and 50-100 mg / mL of LuAG09222.

[0201] A pharmaceutical composition according to any of the above embodiments, comprising E8.100, 150, 200, or 300 mg / ml of eptinezumab.

[0202] A pharmaceutical composition according to any of the above embodiments, comprising LuAG09222 in an amount of E9.50, 75, 100, or 150 mg / ml.

[0203] A pharmaceutical composition according to any one of the above embodiments, comprising E10.100 mg / ml of eptinezumab and 50 mg / ml of LuAG09222.

[0204] E11. The pharmaceutical composition according to any one of the above embodiments, wherein eptinezumab and LuAG09222 are the sole active ingredients in the composition.

[0205] E12. The pharmaceutical composition according to any one of the above embodiments, wherein histidine is present in the composition at a concentration of 10 to 50 mM, optionally 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0206] E13. The pharmaceutical composition according to any one of the above embodiments, wherein polysorbate 80 or poloxamer P188 is present in the composition at a concentration of 0.005 to 0.05% w / v.

[0207] E14. A pharmaceutical composition according to any one of the embodiments described above, wherein one, two, or three of the following excipients, NaCl, sorbitol, and arginine are present in the composition.

[0208] E15. The pharmaceutical composition according to any one of the above embodiments, wherein the concentration of NaCl in the composition is 10 to 150 mM, optionally 25 mM, 50 mM, 100 mM, or 150 mM.

[0209] E16. The pharmaceutical composition according to any one of the above embodiments, wherein sorbitol is present in the composition at a concentration of 50 to 250 mM, optionally 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM.

[0210] E17. The pharmaceutical composition according to any one of the above embodiments, wherein arginine is present in the composition at a concentration of 50 to 250 mM, optionally 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM.

[0211] E18. The pharmaceutical composition according to any one of the embodiments above, wherein the pH is 5.0 to 6.8, optionally pH 5.0, pH 5.5, pH 5.9, pH 6.0, pH 6.5, or pH 6.8.

[0212] E19. The pharmaceutical composition according to any one of the above claims, wherein histidine is in a concentration of 10-50 mM, optionally 20-40 mM, poloxamer P188 is in the range of 0.0025-0.0120% w / v, and polysorbate 80 is in the range of 0.005-0.05% w / v (including both extreme values).

[0213] The pharmaceutical composition according to any one of the above embodiments, wherein the concentration of E20.NaCl is 10 to 150 mM, optionally 30 to 70 mM, the concentration of sorbitol is 50 to 250 mM, optionally 90 to 180 mM, and the concentration of arginine (e.g., L-arginine) is in the range of 50 to 250 mM (including both extreme values).

[0214] A pharmaceutical composition according to any one of the above embodiments, comprising eptinezumab and LuAG90222 in a ratio of E21.2:1, 1:1, or 1:2, and comprising 20-40 mM histidine buffer, 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 pH 5.9).

[0215] A pharmaceutical composition according to any one of the above embodiments, comprising eptinezumab and LuAG90222 in an E22.2:1 ratio, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, 0.0025-0.010% w / v poloxamer P188, and 25-35 mM NaCl, and having a pH of approximately 6 (optionally having a pH of 5.9).

[0216] A pharmaceutical composition according to any one of the above embodiments, comprising eptinezumab and LuAG90222 in an E23.1:1 ratio, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, 0.005-0.010% w / v poloxamer P188, and 45-55 mM NaCl, and having a pH of approximately 6 (optionally pH 5.9).

[0217] A pharmaceutical composition according to any one of the above embodiments, comprising eptinezumab and LuAG90222 in an E24.1:2 ratio, and comprising 35-45 mM histidine buffer, 90-100 mM sorbitol, 0.010-0.0120% w / v poloxamer P188, and 65-75 mM NaCl, and having a pH of approximately 6 (optionally pH 5.9).

[0218] E25. A composition according to any one of the above embodiments for use as a pharmaceutical.

[0219] E26. A composition according to any one of the above embodiments for use in subcutaneous administration.

[0220] E27. A composition according to any one of the above embodiments for use in the treatment or prevention of headache.

[0221] E28. A composition according to any one of the above embodiments for use in the treatment or prevention of pain.

[0222] E29. A composition according to any one of the above embodiments for use in the treatment or prevention of chronic or recurrent migraines.

[0223] E30. A composition according to any one of the above embodiments for use in the treatment or prevention of cluster headaches.

[0224] E31. A composition according to any one of the above embodiments for use in the treatment or prevention of endometriosis.

[0225] E32. 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 flashes, chronic paroxysmal hemiplegia, secondary headache due to underlying structural problems of the head and neck, occipital neuralgia, sinus headache (optionally, e.g., associated with sinusitis), allergic headache or migraine, pain, inflammatory pain, postoperative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, burn pain, osteoporosis, gouty joints The composition according to any one of the above embodiments for use in the treatment or prevention of pain, pain associated with sickle cell attacks and other nociceptive pain, as well as hepatocellular carcinoma, breast cancer, cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, pain due to osteoarthritis or rheumatoid arthritis, lower back pain, diabetic neuropathy, sciatica or gastroesophageal reflux disease, indigestion, irritable bowel syndrome, visceral pain associated with inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis.

[0226] E33. A composition according to any one of the above embodiments for use in the treatment or prevention of chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, eye pain, toothache, postoperative pain, trauma-related pain, diabetes, insulin-independent diabetes and other inflammatory autoimmune diseases, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hypersensitivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, traumatic brain injury, epilepsy, neurodegenerative diseases, skin diseases (such as pruritus, neurogenic skin redness, rosacea and erythema), inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea.

[0227] E34. The composition according to any one of the above embodiments for administration monthly or every two weeks.

[0228] E35. The composition according to any one of the above embodiments, comprising eptinezumab and a fragment of LuAG09222.

[0229] E36. The composition according to Embodiment 26, wherein the eptinezumab and LuAG09222 fragments each comprise all six CDR regions of the antibody.

[0230] E37. The composition according to Embodiment 26, wherein the fragment comprises or consists of eptinezumab and the VH region and VL region of LuAG09222.

[0231] The composition according to any one of the above embodiments, comprising E38.50 to 150 mg / ml, optionally 50 mg / ml, 75 mg / ml, 100 mg / ml, or 150 mg / ml of eptinezumab.

[0232] The composition according to any one of the above embodiments, comprising E39.25 to 100 mg / ml, optionally containing 25 mg / ml, 50 mg / ml, 75 mg / ml, or 100 mg / ml of LuAG09222.

[0233] E40. A composition according to any one of the above embodiments for use in a subcutaneous administration method.

[0234] E41. A method for treating or preventing headache, comprising administering to a patient in need of such treatment one of the compositions described in E1 to E30.

[0235] E42. A method for treating or preventing pain, comprising administering to a patient in need thereof one of the compositions described in any one of E1 to E30.

[0236] E43. A method for treating or preventing chronic or recurrent migraines, comprising administering to a patient in need of such treatment one of the compositions described in E1 to E30.

[0237] E44. A method for treating or preventing cluster headache, comprising administering to a patient in need of such treatment one of the compositions described in E1 to E30.

[0238] E45. A method for treating or preventing endometriosis, comprising administering to a patient in need of such treatment one of the compositions described in E1 to E30.

[0239] E46. 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 flashes, chronic paroxysmal hemiplegia, secondary headache due to underlying structural problems of the head and neck, occipital neuralgia, sinus headache (optionally, e.g., associated with sinusitis), allergic headache or migraine, pain, inflammatory pain, postoperative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, burn pain, osteoporosis, gouty arthralgia, sickle cell attack A method for treating or preventing pain related to or other nociceptive pain, as well as pain associated with or other conditions such as hepatocellular carcinoma, breast cancer, cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, pain due to osteoarthritis or rheumatoid arthritis, lower back pain, diabetic neuropathy, sciatica or gastroesophageal reflux disease, indigestion, irritable bowel syndrome, visceral pain associated with inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis, comprising administering to a patient in need of such treatment or prevention any of the compositions described in any one of E1 to E40.

[0240] E47. A method for treating or preventing chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, eye pain, toothache, postoperative pain, trauma-related pain, diabetes mellitus, non-insulin-dependent diabetes mellitus and other inflammatory autoimmune diseases, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hypersensitivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, traumatic brain injury, epilepsy, neurodegenerative diseases, skin diseases (such as pruritus, neurogenic skin redness, rosacea and erythema), inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea, comprising administering to a patient in need of such treatment or prevention any of the compositions described in any one of E1 to E40.

[0241] Use of the composition according to E1 - E40 for the manufacture of a medicament for the treatment or prevention of headache.

[0242] Use of the composition according to E1 - E40 for the manufacture of a medicament for the treatment or prevention of pain.

[0243] Use of the composition according to E1 - E40 for the manufacture of a medicament for the treatment or prevention of chronic or recurrent migraine.

[0244] Use of the composition according to E1 - E40 for the manufacture of a medicament for the treatment or prevention of cluster headache.

[0245] Use of the composition according to E1 - E40 for the manufacture of a medicament for the treatment or prevention of endometriosis.

[0246] Use of the composition according to E1 - E40 for the manufacture of a medicament for 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, migrainous neuralgia, chronic headache, tension headache, general headache, hot flash, chronic paroxysmal hemicrania, secondary headache due to fundamental structural problems in the head and neck, occipital neuralgia, sinus headache (optionally related to, for example, sinusitis), allergy - induced headache or migraine, pain, inflammatory pain, postoperative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain due to burns, osteoporosis, gouty arthritis pain, pain associated with sickle cell crisis and other nociceptive pain and hepatocellular carcinoma, breast cancer, cirrhosis, neuropathic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, post - herpetic neuralgia, phantom limb pain, fibromyalgia, dysmenorrhea, ovarian pain, reflex sympathetic dystrophy, neuropathic pain, pain due to osteoarthritis or rheumatoid arthritis, low back pain, diabetic neuropathy, sciatica or gastroesophageal reflux disease, dyspepsia, irritable bowel syndrome, visceral pain associated with inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis.

[0247] E54. Use of compositions described in E1 to E40 for the manufacture of pharmaceuticals for the treatment or prevention of chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, eye pain, toothache, postoperative pain, trauma-related pain, diabetes, insulin-independent diabetes and other inflammatory autoimmune diseases, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hypersensitivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, traumatic brain injury, epilepsy, neurodegenerative diseases, skin diseases (such as pruritus, neurogenic skin redness, rosacea and erythema), inflammatory bowel disease, irritable bowel syndrome, cystitis and dysmenorrhea.

[0248] E55. Eptinezumab is derived from the following 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 A composition, method, or use according to any of the above embodiments, comprising all of the above.

[0249] E56.LU AG09222 consists of the following six CDR sequences: Light chain CDR1 SEQ ID NO: 17, Light chain CDR2 SEQ ID NO: 18, Light chain CDR3 SEQ ID NO: 19, Heavy chain CDR1 SEQ ID NO: 12, Heavy chain CDR2 SEQ ID NO: 13, and Heavy chain CDR3 SEQ ID NO: 14 A composition, method, or use according to any of the above embodiments, comprising all of the above.

[0250] E57. The composition, method, or use according to any of the above embodiments, wherein eptinezumab has the VH region described in SEQ ID NO: 4 and the VL region described in SEQ ID NO: 10.

[0251] E58.LU AG09222 is a composition, method, or use according to any of the above embodiments, having a VH region as described in Sequence ID No. 15 and a VL region as described in Sequence ID No. 20.

[0252] E59. The composition, method, or use according to any of the above embodiments, wherein eptinezumab has a heavy chain as described in SEQ ID NO: 5 or SEQ ID NO: 6 and a light chain as described in SEQ ID NO: 11.

[0253] E60.LU AG09222 is a composition, method, or use according to any of the above embodiments, comprising a heavy chain described in Sequence ID No. 16 or Sequence ID No. 26 and a light chain described in Sequence ID No. 21.

[0254] E61. The composition, method, or use according to any of the above 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.

[0255] Additional exemplary embodiments (EE) Embodiment 1 (EE1) A method of treating a patient in need by administering an effective dose of eptinezumab and LuAG09222.

[0256] EE2. The method according to EE1, in which eptinezumab and LuAG09222 are administered simultaneously or sequentially.

[0257] EE3. The method according to any one of the above embodiments, wherein eptinezumab and LuAG09222 are administered sequentially within a period of 0.5 to 2 hours, for example, within 0.5 hours or 1 hour.

[0258] The method according to any one of the above embodiments, wherein EE4.50-150 mg / ml of eptinezumab is administered, optionally at doses of 50 mg / ml, 75 mg / ml, 100 mg / ml, or 150 mg / ml.

[0259] The method according to any one of the above embodiments, wherein LuAG09222 is administered at an EE of 5.25 to 100 mg / ml, with optional doses of 25 mg / ml, 50 mg / ml, 75 mg / ml, or 100 mg / ml.

[0260] EE6. The method according to any one of the above embodiments, wherein eptinezumab is administered subcutaneously or intravenously, and LuAG09222 is administered subcutaneously or intravenously.

[0261] EE7. The method according to any one of the above embodiments for treating or preventing headache.

[0262] EE8. The method according to any one of the above embodiments for treating or preventing pain.

[0263] EE9. The method according to any one of the above embodiments for treating or preventing chronic or recurrent migraine.

[0264] EE10. The method according to any one of the above embodiments for treating or preventing cluster headache.

[0265] EE11. The method according to any one of the above embodiments for treating or preventing endometriosis.

[0266] EE12. 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 flashes, chronic paroxysmal hemiplegia, secondary headache due to underlying structural problems of the head and neck, occipital neuralgia, sinus headache (optionally, e.g., associated with sinusitis), allergic headache or migraine, pain, inflammatory pain, postoperative incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, burn pain, osteoporosis, gout The method according to any one of the above embodiments for treating or preventing sickle cell carcinoma, pain associated with sickle cell attacks and other nociceptive pain, as well as hepatocellular carcinoma, breast cancer, cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, pain due to osteoarthritis or rheumatoid arthritis, lower back pain, diabetic neuropathy, sciatica or gastroesophageal reflux disease, indigestion, irritable bowel syndrome, visceral pain associated with inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, childbirth, menopause, prostatitis or pancreatitis.

[0267] EE13. The method according to any one of the above embodiments for treating or preventing chronic pain, neurogenic inflammation and inflammatory pain, neuropathic pain, eye pain, toothache, postoperative pain, trauma-related pain, diabetes mellitus, non-insulin-dependent diabetes mellitus and other inflammatory autoimmune diseases, vascular disorders, inflammation, arthritis, sarcoidosis, bronchial hypersensitivity, asthma, shock, sepsis, opioid withdrawal syndrome, morphine tolerance, hot flashes in men and women, allergic dermatitis, psoriasis, encephalitis, traumatic brain injury, epilepsy, neurodegenerative diseases, skin diseases (such as pruritus, neurogenic skin redness, rosacea and erythema), inflammatory bowel disease, irritable bowel syndrome, cystitis, and dysmenorrhea.

[0268] EE14. The method according to any one of the above embodiments, wherein eptinezumab and LuAG09222 are administered monthly (every four weeks) or every two weeks.

[0269] EE15. Eptinezumab is derived from the following 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 A method according to any one of the above embodiments, comprising all of the above.

[0270] EE16.LUAG09222 consists of the following six CDR sequences: Light chain CDR1 SEQ ID NO: 17, Light chain CDR2 SEQ ID NO: 18, Light chain CDR3 SEQ ID NO: 19, Heavy chain CDR1 SEQ ID NO: 12, Heavy chain CDR2 SEQ ID NO: 13, and Heavy chain CDR3 SEQ ID NO: 14 A method according to any one of the above embodiments, comprising all of the above.

[0271] EE17. The method according to any one of the above embodiments, wherein eptinezumab has a VH region as described in SEQ ID NO: 4 and a VL region as described in SEQ ID NO: 10.

[0272] EE18.LU AG09222 is the method according to any one of the above embodiments, having a VH region described in Sequence ID No. 15 and a VL region described in Sequence ID No. 20.

[0273] EE19. The method according to any one of the above embodiments, wherein eptinezumab has a heavy chain as described in SEQ ID NO: 5 or SEQ ID NO: 6 and a light chain as described in SEQ ID NO: 11.

[0274] EE20.LU AG09222 is a method according to any one of the above embodiments, comprising a heavy chain described in Sequence ID No. 16 or Sequence ID No. 26 and a light chain described in Sequence ID No. 21.

[0275] The above description of various exemplary embodiments of the present invention is not intended to be exhaustive or to limit the invention to the exact forms disclosed. While specific embodiments and examples of the present invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as will be apparent to those skilled in the art. The teachings of the present invention provided herein can be applied to purposes other than those described above.

[0276] In light of the detailed description above, these and other modifications to the present invention may be made. In general, the terms used in the following claims should not be construed as limiting the invention to any specific embodiment disclosed herein and in the claims. Accordingly, the invention is not limited by this disclosure, and instead, the scope of the invention should be entirely determined by the following claims.

[0277] The present invention may be carried out in ways other than those specifically described in the foregoing description and examples. In light of the above teachings, numerous modifications and variations of the present invention are possible and therefore fall within the scope of the appended claims.

[0278] The entire disclosures of each document cited in the Background, Detailed Description and Examples of the Invention (including patents, patent applications, journal articles, abstracts, manuals, books, or other disclosures) are incorporated herein by reference in their entirety.

[0279] The following examples are provided to give a detailed disclosure and explanation of how the present invention is prepared and used, and are not intended to limit the scope of what is considered the present invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, concentration, etc.), but some experimental error and deviation should be acceptable. Unless otherwise specified, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near thereto. [Examples]

[0280] The following embodiments are provided to illustrate the present invention and should not be construed as limiting the scope of the claims.

[0281] Example 1 Human clinical trials to evaluate the safety and efficacy of the anti-CGRP antibody according to the present invention. Clinical treatment protocols The humanized anti-CGRP IgG1 antibody, identified herein as Ab6 (eptinezumab), was evaluated in human subjects for its ability to inhibit, reduce, or prevent the number, duration, and / or severity of migraine episodes.

[0282] Specifically, the clinical efficacy of Ab6 antibodies was investigated in a placebo-controlled, double-blind, randomized trial. All participants in the trial were selected based on specific criteria. In particular, all were ≤50 years old, diagnosed with migraine (ICHD-II, 2004 Section 1), and had a history of migraine for ≥12 months with ≥5 and ≤14 migraine days in 28 days each during the 3 months prior to screening.

[0283] Furthermore, during the three months prior to screening and the 28 days prior to the end of the e-diary before randomization, all individuals participating in the study used acute migraine medication on ≤14 days per 28-day period, and within those days, they used triptans on ≤10 days per 28-day period.

[0284] Table 1 summarizes the demographic characteristics of the test population.

[0285] [Table 2]

[0286] Throughout the study, all participants were required to record their migraine condition daily using an e-diary. The e-diary required participants to record the number of migraine days / month, migraine episodes / month, migraine duration / month, migraine severity, and the use of any preventative medications, including triptans.

[0287] In addition, trial participants were asked to use an e-diary to record their migraine status for 28 days prior to antibody or placebo treatment, establishing a monthly migraine day / hour / episode baseline. This also allowed the trial participants to become familiar with using the e-diary.

[0288] After 28 days of the experiment, the subjects were divided into two groups, each containing 80 subjects (Figure 5). In the first group, the antibody treatment group (n=80), each subject received a single 1000 mg dose of Ab6 intravenously. In the second group (n=80), the placebo group, each subject received an intravenous injection containing only an antibody carrier aqueous solution.

[0289] Individuals in the treatment and placebo groups were evaluated 24 weeks after dose administration. First, a 12-week interim analysis was performed. Following the 12-week interim analysis, a refined analysis was conducted. This refined analysis potentially involved, for example, the addition or removal of patient data according to the study protocol, or updating data that was not fully loaded from e-diaries. This refinement resulted in minor changes but did not alter the overall conclusions.

[0290] The efficacy of the antibody compared to placebo was evaluated in part based on data recorded in e-diaries. For example, this analysis included a comparison of the number of recorded migraine days / month, migraine episodes / month, and migraine duration / month in the treatment group and the placebo group. The proportion of responders in each group (i.e., subjects who experienced a 50%, 75%, and 100% reduction in migraine days) was also compared.

[0291] In addition, responses to the MSQ and HIT-6 questionnaires in both groups for Ab6 and placebo will be evaluated and compared. The MSQ is a disease-specific tool frequently used to assess the impact of migraine on health-related quality of life (HRQL). The MSQ includes a 16-item migraine-specific quality of life questionnaire (version 1.0) developed by Glaxo Wellcome Inc. The MSQ is assumed to measure three parameters: (i) role function limitation, (ii) role function prevention, and (iii) emotional function.

[0292] The HIT-6, or Functional Impact Test (also known as the Headache Impact Test or HIT-6), is a well-known tool for assessing migraine severity. This test uses six questions to understand the impact of headaches and their treatment on an individual's functional health and well-being.

[0293] Clinical results and analysis The results of this 12-week human clinical trial and analysis in the treatment population are summarized in Table 2 below.

[0294] [Table 3]

[0295] In addition, the results of the clinical trials were compared based on the number of responders in the treatment and placebo groups. As shown in Figure 1, the number of subjects who showed a 50%, 75%, or 100% reduction in migraine days over each month of the interim period was compared between the treatment and placebo groups. As shown in the figure, 60% of the Ab6 treatment group experienced at least a 50% reduction in headache days, 31% of the Ab6 treatment group experienced at least a 75% reduction in headache days, and 15% of the Ab6 treatment group experienced a 100% reduction in headache days.

[0296] In contrast, 33% of the placebo group experienced a reduction of at least 50% in headache days, 9% of the placebo group experienced a reduction of at least 75%, and 0% of the placebo group (no treatment) experienced a 100% reduction in headache days.

[0297] These results clearly demonstrate a much greater reduction in migraine days in the Ab6 treatment group. Without a significant placebo effect, these differences would have been even more pronounced. (The increased placebo effect is not surprising, as this phenomenon is often very pronounced with migraine and other neurological medications.)

[0298] In addition, the percentage change from baseline in the number of migraine days per month in the placebo group and the Ab6 treatment group was compared. As shown in Figure 2, the median (±QR)% change from baseline in the number of migraine days per month in the placebo group and the Ab6 treatment group was compared for the two groups over 12 weeks after treatment. These results were statistically significant (p=0.0078), clearly indicating that the Ab6 treatment group experienced a significantly greater reduction in the number of headache days per month compared to baseline than the placebo group.

[0299] The percentage change from baseline in the number of migraine episodes per month in the placebo group and the Ab6 treatment group was also compared. As shown in Figure 3, the median (±QR)% change from baseline in the number of migraine episodes per month in the placebo group and the Ab6 treatment group was compared over 12 weeks after treatment. These results indicate that the number of migraine episodes per month in the Ab6 treatment group was significantly higher than that in the placebo group compared to baseline.

[0300] Furthermore, the percentage change from baseline in monthly migraine episode duration was compared between the placebo group and the Ab6 treatment group. As shown in Figure 4, the median (±QR)% change from baseline in monthly migraine duration was compared for both groups over 12 weeks after treatment. These results indicate that the Ab6 treatment group experienced a significantly greater reduction in the number of monthly migraine episodes compared to baseline than the placebo group.

[0301] In addition, HIT-6 results were compared between the two groups. As mentioned above, this questionnaire is a well-accepted method for assessing the migraine status of individuals with frequent / chronic migraines. Figure 6 compares HIT-6 responder analyses for the Ab6 treatment group and the placebo group at baseline, 4 weeks post-treatment, 8 weeks post-treatment, and 12 weeks post-treatment. Results at each time point showed that the Ab6 treatment group had a statistically significant improvement in HIT-6 scores compared to the placebo group: 54.4% in the Ab6 treatment group compared to 30% in the placebo group at 4 weeks (p=0.0023), 51.3% in the Ab6 treatment group compared to 38.0% in the placebo group at 8 weeks (p=0.1094), and 61.1% in the Ab6 treatment group compared to 33.3% in the placebo group at 12 weeks (p=0.0007). Figure 7 shows the proportion of patients with some or little / no HIG-6 scores over time in the placebo and Ab6 treatment groups (statistical significance is shown).

[0302] In addition, Figure 8 shows the pharmacokinetic (PK) profile (in mg / mL) of Ab6 administered intravenously at a single dose of 1000 mg over a 24-week period following Ab6 administration.

[0303] Figure 9 shows the plasma-free pharmacokinetic (PK) parameters N (number of patients), mean, and standard deviation (SD) for a single 1000 mg intravenous dose of Ab6. The parameters and units shown in the table are C. max (μg / mL), AUC 0-∞ (mg*hr / mL), Half-life (days), V z (L) and C L (mL / hr)

[0304] Further analysis was conducted on patient data from weeks 12 to 24. The treatment group continued to show a reduction in the number of migraine days compared to the control group, although the magnitude of this difference decreased over time. In addition, the control group had fewer migraine days per month than baseline. This was thought to be at least partly due to "diary fatigue," where patients may not report migraines on the days they actually occurred in order to avoid the time and effort required to answer further questions about migraines that would arise from giving a positive answer to the question of whether they had a migraine on a given day.

[0305] Further analysis of the trial results is shown in Figures 10–21. These results include an analysis of the change from baseline (mean + / SEM) in the number of migraine days per month for Ab6 (1000 mg iv) versus placebo (Figure 22), and the change in the mean number of migraine days (+ / SD) over time for the entire analysis population (Figure 23). In addition, the distribution of actual migraine days and changes for the Ab6 treatment group during weeks 1–4 (Figure 12), the distribution of actual migraine days and changes for the placebo group during weeks 1–4 (Figure 13), the distribution of actual migraine days and changes for the Ab6 treatment group during weeks 5–8 (Figure 14), the distribution of actual migraine days and changes for the placebo group during weeks 5–8 (Figure 15), the distribution of actual migraine days and changes for the Ab6 treatment group during weeks 9–12 (Figure 16), and the distribution of actual migraine days and changes for the placebo group during weeks 9–12 (Figure 17).

[0306] Responder rate analysis was also performed (Figures 18-20). These figures show the 50%, 75%, and 100% responder rates for the Ab6 and placebo treatment groups, respectively. Subjects with a 50% or greater reduction in migraine frequency were considered 50% responders. Subjects with a 75% or greater reduction in migraine frequency were considered 75% responders. Similarly, subjects with a 100% reduction in migraine frequency were considered 100% responders.

[0307] In Figures 10 and 18-20, the e-diary for days 21-27 was completed by applying normalization to the visit interval and multiplying the observed frequency by the reciprocal of the completion rate.

[0308] Migraine severity was also analyzed. Figure 21 shows the time-averaged migraine severity for the entire analysis group. On the scale used, a mean migraine score of 3 represents "moderate headache."

[0309] Figure 22 summarizes the changes from baseline in outcome scales including migraine days, migraine episodes, migraine duration, mean migraine severity, headache frequency, and HIT-6 score, MSQ (Migraine-Specific Quality of Life Questionnaire) RFP (Role Functioning Prevention), MSQ RFR (Role Functioning Limitation), and MSQ EF (Emotional Functioning).

[0310] Example 2 Human clinical trials to evaluate the safety and efficacy of anti-CGRP antibodies in patients with chronic migraine. This example describes a randomized, double-blind, placebo-controlled clinical trial evaluating the safety and efficacy of Ab6 (eptinezumab) for the prevention of chronic migraine. In this trial, 1,072 patients were randomized to receive either Ab6 (300 mg or 100 mg) or placebo administered by infusion every 12 weeks. To be eligible for this trial, patients had to experience at least 15 days of headache per month, with at least 8 of those days being migraines. Patients participating in the trial had a mean 16.1 days of migraine per month at baseline. Trial endpoints included the mean change from baseline in monthly migraine days, reduction in migraine prevalence on day 1 and days 1–28, and at least 50%, 75%, and 100% reductions from baseline in mean monthly migraine days, change from baseline in mean monthly acute migraine-specific medication days, and reduction from baseline in patient-reported impact scores for the Headache Impact Test (HIT-6).

[0311] Patient characteristics are summarized in Figure 27, with separate columns for patients receiving placebo, 100 mg antibody, or 300 mg antibody. The mean years since migraine diagnosis were 17.0–19.0 ​​years, the mean duration of chronic migraine was 11.5–12.4 years, and 44.3–45.2% of patients were using at least one preventive medication. At baseline, the mean number of migraine days per month was 16.1 in both antibody treatment groups, compared to 16.2 in the placebo group.

[0312] A specific percentage (50%, 75%, or 100%) reduction in average monthly migraine days from baseline refers to the number or proportion of patients in the treatment group who showed a given percentage reduction in monthly migraine days. For example, a patient who had 16 migraine days per month at baseline would be a 75% responder if their monthly migraine days decreased by at least 12 days per month over a specific period.

[0313] The results are shown in Figures 23-27. Figure 23 shows the percentage of patients with migraines in the 300 mg, 100 mg, and placebo treatment groups on days 1, 7, 14, 21, and 28. The top line represents the placebo result, the bottom line represents the 300 mg dose result, and the middle line represents the 100 mg dose result.

[0314] As shown in Figure 23, on day 1, the reduction in migraine prevalence was 52% for the 300 mg dose, 50% for the 100 mg dose, and 27% for the placebo group. This reduction was statistically significant compared to placebo in both the 100 mg and 300 mg treatment groups.

[0315] Figures 24-26 show the percentage of patients in the 300 mg and 100 mg treatment groups who achieved a 50%, 75%, and 100% reduction in migraine days, respectively, at 1 month, 1-3 months (after the first infusion), and 4-5 months (after the second infusion). In each graph, the data bars show the results for the 100 mg, 300 mg, and placebo groups from left to right. Statistical significance is as indicated. ++ indicates statistical significance compared to placebo, + indicates statistical significance compared to placebo (unadjusted), and § indicates statistical significance compared to placebo (post-hoc).

[0316] Example 3 Baseline subgroup analysis for human clinical trials evaluating the safety and efficacy of anti-CGRP antibodies in patients with chronic or sudden migraine. In the chronic migraine trial described in Example 3, each patient was evaluated for potential drug overuse headache (MOH) at the time of administration. MOH was observed in 39.9% (139 patients) of the 100 mg treatment group, 42.0% (147 patients) of the 300 mg treatment group, and 39.6% (145 patients) of the placebo group. Evaluation of treatment outcomes in this patient subset showed that treatment with anti-CGRP antibodies was effective for MOH (Figure 29). Specifically, in the 100 mg treatment group, the mean monthly migraine days per patient with MOH at baseline changed by -3.0 days (95% CI, -4.56 to -1.52 days) compared to patients with MOH who received placebo. Similarly, in the 300 mg treatment group, the mean monthly migraine days per patient with MOH at baseline changed by -3.2 days (95% CI, -4.66 to -1.78 days) compared to patients with MOH who received placebo. In contrast, among patients who did not have MOH at baseline, the 100 mg treatment group showed a mean change of -1.3 days per month (95% CI, -2.43 to -0.16 days) compared to patients who did not have MOH at baseline and received placebo. Similarly, among patients who did not have MOH at baseline, the 300 mg treatment group showed a mean change of -2.1 days per month (95% CI, -3.24 to -0.88 days) compared to patients who did not have MOH at baseline and received placebo. Efficacy was also demonstrated in other subgroups, including patients with a mean migraine duration (MMD) frequency of less than 17 days or 17 days or more; patients aged 21 years or younger or over 21 years at diagnosis; patients with a migraine duration of 15 years or less or over 15 years; patients with migraines with or without aura; patients with or without prior prophylactic medication use; patients with or without concomitant prophylactic medication use; and patients with 33% or more or less triptan use days. Efficacy for each subgroup is shown in each case (Figure 29).

[0317] In another human clinical trial of patients with sudden migraine, in a double-blind, parallel study, patients were randomized to receive Ab6 100 mg (n=221), 300 mg (n=222), or placebo (n=222). After a 28-day screening period, patients received intravenous administration of the drug or placebo every 3 months for a total of four infusions (Figure 28). Efficacy was demonstrated over 1–3 months for both the 100 mg and 300 mg treatment groups, with a mean change in migraine days of -3.9 days in the 100 mg treatment group and -4.3 days in the 300 mg treatment group, compared to -3.2 days in the placebo group. The study also demonstrated efficacy in a subset of patients, including those with a mean migraine duration (MMD) frequency of 9 days or less or more than 9 days, those aged 21 years or younger or over 21 years at diagnosis, those with a migraine duration of 15 years or less or over 15 years, and those with migraines with or without aura.

[0318] Example 4 The effect of Ab6 treatment on drug use in patients with chronic and sudden migraines. During the studies of the chronic migraine patient described in Example 3 and the idiopathic migraine patient described in Example 4, patients were allowed to use acute medications at their own discretion and also recorded their use of acute medications in their daily e-diary. Acute medications for migraines included ergot, triptans, and analgesics (e.g., NSAIDs, opioids, and caffeine-containing combination analgesics).

[0319] For further analysis, patients were stratified by the number of days they used acute drugs during a 28-day screening period (1–9 or ≥10 days, "baseline"). The number of acute drug days was calculated for each individual type of acute drug; combinations, i.e., when two or more types of drugs were used on the same calendar day, were counted as separate drug use days. For example, if a patient took an opioid and a triptan on the same day, it was counted as two days of acute drug use. These analyses included patients who had at least one day of acute drug use during the 28-day baseline screening period.

[0320] In both chronic and sudden-onset migraine patients using acute medication during a 28-day baseline period, Ab6 treatment resulted in a greater average reduction in monthly migraine and acute medication days compared to placebo, as early as one month after administration, with similar results obtained over two dose intervals over six months.

[0321] Ab6 consistently demonstrated a significant reduction in the average number of migraine days per month over the 6-month treatment period compared to placebo in chronic migraine patients who had at least one day of acute medication use at baseline (Figure 30). Chronic migraine patients with at least one day of acute medication use per month at baseline showed a significant reduction in acute medication use compared to placebo, both as early as one month after treatment and throughout the entire treatment period (Figure 31). In a subset of chronic migraine patients who had taken acute medication for 1–9 days at baseline, the change from baseline in the number of days of acute medication use was greater in the 300 mg Ab6 group than in placebo over the 6-month treatment period (Figure 32). In patients with at least 10 days of medication use at baseline, a clear reduction in the number of days of medication per month was observed in both Ab6 treatment groups compared to racebo over the entire 6-month period. Figure 33 shows the change in medication use at 1 month and 6 months in subsets of chronic migraine patients with 1 or more days, 1–9 days, and 10 or more days of acute medication use at baseline. Except for Ab6 100 mg at 6 months in patients using 1–9 days / month at baseline, Ab6 showed a greater therapeutic effect than placebo in reducing acute drug use.

[0322] Similarly, patients with idiopathic migraine who had at least one day of acute medication use during baseline over two dose intervals spanning six months experienced a significantly greater reduction in the mean number of migraine days per month with Ab6 compared to placebo (Figure 34). Patients with idiopathic migraine who had at least one day of acute medication use per month during baseline showed a significantly greater reduction in acute medication use than placebo, both as early as one month after treatment and throughout the entire treatment period (Figure 35). In a subgroup of patients with idiopathic migraine who had taken acute medication for 1–9 days during baseline, the change from baseline in the number of days of acute medication use was greater with Ab6 than with placebo over the six months of treatment (Figure 36). A similar pattern was observed in a subgroup of patients who had taken acute medication for 10 days or more during baseline, but the small sample size may have contributed to the less consistent pattern over time. Figure 37 shows the changes in medication use at 1 month and 6 months in a subset of patients with sudden migraine who had acute medication use for 1 day or more, 1 to 9 days, or 10 days or more at baseline. Except for Ab6 100 mg at 6 months in patients who had used medication for 10 days / month or more at baseline, the reduction in acute medication use was greater in the Ab6 treatment group than in the placebo group.

[0323] These results indicate that both patients with sudden-onset and chronic migraines who were at risk of medication overuse headache (≥10 days / month of acute medication use) showed the greatest reduction in acute medication use, suggesting that Ab6 therapy generally resulted in a greater reduction in medication use days than placebo.

[0324] The most frequently reported acute headache medications in over 10% of the subjects were tomapirin N (44.5%) (a combination of paracetamol, aspirin, and caffeine), ibuprofen (40.6%), sumatriptan (33.6%), paracetamol (acetaminophen) (20.3%), and naproxen sodium (10.2%). The most frequently reported prophylactic headache medication in over 10% of the subjects was topiramate (12.5%).

[0325] Example 5 Efficacy of anti-CGRP antibodies in patients experiencing acute migraine attacks This example describes a randomized, double-blind, placebo-controlled clinical trial evaluating the safety and efficacy of Ab6 for the acute treatment of migraines. In this trial, approximately 450 patients will be randomized in a 1:1 ratio to receive either 100 mg of Ab6 or placebo. During the screening period (approximately 1–8 weeks), patients will be assessed for migraine frequency and medication use frequency. Eligible patients will have a migraine attack frequency of approximately 4–15 migraine days per month for the three months prior to screening. Based on medical history, a typical migraine attack in a subject, if left untreated, would involve moderate to severe headache and the most troublesome symptoms of nausea, photophobia, or phonophobia. Subjects must be headache-free for at least 24 hours prior to the onset of a migraine in order to participate in the trial. On the day of treatment, patients will go to the study site, and intravenous injection of 100 mg of Ab6 or placebo will be initiated approximately 1–6 hours after the onset of an attack. The patient had not received any other monoclonal antibody (e.g., any CGRP antagonist antibody) within the six months prior to screening.

[0326] The co-primary endpoints are time to headache resolution and time to resolution of the most distressing symptom. Important co-secondary endpoints are absence of headache at 2 hours and absence of the most distressing symptom at 2 hours. Secondary endpoints include time to headache resolution, absence of headache at 2 hours and absence of persistent headache over 24 and 48 hours, use of emergency medication up to 24 and 48 hours, absence of photophobia at 2 hours, absence of phonophobia at 2 hours, absence of nausea at 2 hours, change from baseline in the Headache Impact Test (HIT 6) at 4 weeks, and change from baseline in the Migraine Treatment Optimization Questionnaire-6 (mTOQ-6) at 4 weeks. Exploratory endpoints include the absence of headache at all time points except 2 hours, the absence of photophobia at all time points except 2 hours, the absence of phonophobia at all time points except 2 hours, the absence of nausea at all time points except 2 hours, pain recurrence if the subject had no headache at 2 hours, the patient's general impression change (PGIC) at 4 weeks, and time to the next migraine. Headache was collected on a 4-point scale, with 3 being severe, 2 being moderate, 1 being mild, and 0 being painless. The absence of pain was defined as no pain (0) without the use of emergency medication (it should be noted that in the study, emergency medication should not be used for 2 hours after the completion of infusion to decouple the effect of the antibody from the emergency medication, but emergency medication may be used selectively in the course of normal use, and any use of emergency medication will be collected as data).

[0327] Statistical analysis is performed to determine the significance of the differences in endpoints between patients receiving Ab6 or placebo, including time to pain relief, time to relief of the most troublesome symptoms, and each of the other aforementioned endpoints.

[0328] The use of emergency medication refers to any therapeutic intervention (medical or device) given to a subject to provide relief of migraines. In this study, emergency medication is not prohibited, although it should not be provided earlier than two hours after completion of the study drug administration in order to decouple the effects of the antibody from the effects of the emergency medication. The proportion of subjects requiring the use of emergency medication will be summarized in this study. Acute emergency medication includes any drug for treating migraines or migraine-related symptoms, such as triptans, analgesics, such as non-opioids or opioid / anesthetics, acetaminophen, NSAIDs, combination drugs, such as EXCEDRIN® or EXCEDRIN MIGRAINE®, antiemetics, ergotamine, ergot derivatives, etc.

[0329] The absence of migraine-related symptoms (photophobia, phonophobia, and nausea) refers to the absence or presence of each of the aforementioned migraine-related symptoms reported by the subjects. The percentage of subjects who were asymptomatic without the administration of emergency medication is summarized in this study.

[0330] The Headache Impact Test (HIT-6) is evaluated as a change from baseline in the total score, which is then aggregated and compared between treatment groups in this study.

[0331] The Migraine Treatment Optimization Questionnaire-6 (mTOQ-6) will be assessed as a change from baseline in the total score, aggregated in this study, and compared between treatment groups.

[0332] The time to headache relief is assessed as the first point after completion of the infusion when the subject reports pain relief, meaning that the subject's headache changed from moderate or severe (2 or 3) to mild or painless (1 or 0) without the administration of emergency medication.

[0333] Pain recurrence is assessed as the occurrence of headache of any severity within 48 hours of drug administration in patients who were headache-free (0) at 2 hours. The proportion of subjects who experienced headache recurrence of any severity will be summarized in this study.

[0334] This trial demonstrates that Ab6 is effective and safe for treating acute migraines.

[0335] Example 6 In pivotal clinical trials, patients were administered Ab6 in doses of 100 mg or 300 mg, as described in Example 2. Including day -1 (after Ab6 infusion) in the statistical analysis demonstrates that a clear therapeutic effect exists immediately after infusion when the therapeutic effect is evaluated (Figure 38). In the figure, day 0 is defined as the infusion day, and the data for day -1 represents the pre-infusion condition. A significant reduction in the percentage of migraines from day -1 (baseline, the day before infusion) to day 0 is evident. Furthermore, the magnitude of the effect is higher with the 300 mg dose than with the 100 mg dose, and both doses show a higher effect than the placebo group.

[0336] Example 7 This example concerns the antibody Ab10.H3 (LuAG09222). Ab10.H3 has been studied in multiple clinical trials, including a Phase I trial to determine the safety and tolerability of escalating doses, and has recently demonstrated migraine prevention efficacy in a Phase II trial (the aforementioned HOPE trial). Thus, Ab10.H3 has been proven to be safe and useful in the treatment of migraine patients.

[0337] Ab10.H3 is described in International Patent Application Publication No. 2017181039, and its contents, including the sequence listing, are incorporated herein by reference in their entirety. Ab10.H3 is a humanized version of the antibody Ab10 described in International Patent Application Publication No. 2017181039. International Patent Application Publication No. 2017181039 describes that Ab10 and AB10.H3 have higher inhibitory specificity against the PCAP1 receptor pathway than against the VPAC1 or VPAC2 pathway. In the case of Ab10, humanization of Ab10 resulted in a decrease in IC50(pM) for PACAP38-induced PCA1-R-mediated cAMP increase, but humanization resulted in an increase in IC50(pM) for PACAP38-induced VPAC1-R-mediated and VPAC2-mediated cAMP increase, which indicates an overall increase in inhibitory specificity against the PCAP1 receptor. Considering the abundant expression of VCAP1 and VCAP2 receptors outside the nervous system (see paragraph

[0005] of International Publication No. 2017181039), a decrease in relative specificity to VCAP1 and VCAP2 receptors is considered desirable because it reduces unintended effects, i.e., unwanted interactions with target cells, when the antibody is used as a therapeutic or prophylactic agent.

[0338] Therefore, humanized antibodies offer additional technical features, namely improved inhibitory specificity of the PCAP1 receptor pathway to the VPAC1 or VPAC2 pathway, which further complements the aforementioned technical differences. The following table summarizes these points from International Publication No. 2017181039. Table 2 is based on a table cited from International Publication No. 2017181039 and was compiled from International Publication No. 2017181039.

[0339] [Table 4]

[0340] The Ab10 and AB10.H3 antibodies exhibit neurological effects in vivo: As demonstrated in Example 11 of International Publication No. 2017181039, Ab10.H and Ab10.H3 reduce photosensitivity in a mouse model of PACAP-induced photosensitivity (see, for example, paragraph

[0956] of International Publication No. 2017181039). Since photosensitivity, i.e., extreme photosensitivity, is a symptom commonly experienced by patients with neurological disorders such as migraines, the antibodies claimed herein are antibodies that can be used for the treatment or prevention of such neurological disorders or symptoms. In Example 13 of International Publication No. 2017181039, Ab10.H3 reduced the trigeminal parasympathetic reflex in a rat model of vascular dysfunction for cluster headache, trigeminal neuralgia, and occasionally migraine, as measured by tear secretion volume and nasal temperature after intranasal administration of umberlon (see paragraphs

[0967] to

[0972] and figures 34 and 35 of International Publication No. 2017181039). Furthermore, Example 8 of International Publication No. 2017181039 shows that Ab10 inhibits PACAP-induced cutaneous vasodilation in rabbits. While this experiment examined cutaneous vasoactive effects, the neural mechanisms of vasoconstriction / vasodilation are almost identical in the brain, and vasodilation is one of the triggering mechanisms for various headaches. In summary, these antibodies may be used for the treatment or prevention of neurological diseases or symptoms such as headaches.

[0341] Ab10 and AB10.H3 recognize unique epitopes within PACAP: As demonstrated in Example 12 of International Publication No. 2017181039, Ab10 and Ab10.H3 recognize residues 19, 22, 23, and 27 of PACAP. The humanized version of Ab10 claimed herein is also expected to recognize the same residues, as it shares the same six CDR sequences as the parent antibody Ab10. Binding to these specific epitopes, particularly residues 23 and 27, is expected to confer the unique and beneficial feature described herein, which is the inability to bind to PAC1 receptor-expressing cells via PACAP.

[0342] Affinity: Humanization of Ab10 increased its 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 in International Publication No. 2017181039).

[0343] IC50: Humanization of Ab10 reduced the IC50 (pM) for PACAP38-induced PCA1-R-mediated cAMP increase from 180.3 to 163.4 (Ab10.H), 21.3 (Ab10.H2), 30.7 (Ab10.H3), 22.8 (Ab10.H4), and 22.7 (Ab10.H5) pM (see Tables 2 and 3 of International Publication No. 2017181039).

[0344] Example 7 The high-concentration co-formulation eptinezumab LuAG00922 requires a delicate balance of formulation components and pH. The correct balance of components and pH results in low viscosity, enabling subcutaneous administration and achieving a stable formulation for long-term storage. In typical monoclonal antibody (mAb) formulations, steric and colloidal stability are optimized based on the unique biophysical properties of the specific antibody. In the case of a co-formulation of two mAbs, formulation development becomes difficult if the biophysical properties of each mAb are distinctly different. In this case, the isoelectric point of eptinezumab is approximately 8.1, while that of LuAG00922 is approximately 6.9. Often, to improve colloidal stability and solubility, which are key to high-concentration stability, it is desirable to formulate mAbs with a composition that differs from the isoelectric point by several pH units. Furthermore, the behavior of LuAG00922 as the sole antibody component in high-concentration formulations exhibits a unique pH-dependent viscosity. When the concentration of LuAG00922 exceeds 158 mg / mL at pH 5.5, the LuAG00922 formulation exhibits a relatively high viscosity >70 cP. As the pH increases from 5.5 to 6.0 and further to 6.5, the viscosity of LuAG00922 shows a pH-dependent charge patch, changing from a protonated positively charged patch at pH 5.5 to a deprotonated neutrally charged patch at pH 6.0 and 6.5, with viscosity decreasing as the pH increases.

[0345] Design of Experiments (DOE) The formulation design space was constructed by varying the pH and the amounts of L-histidine, sorbitol, L-arginine hydrochloride, poloxamer 188, and protein concentrations. A pH range of 5.25–6.75 was explored, as this was desired to impart buffering capacity to the mAb subcutaneous formulation, given the desired pH range using 10–50 mM histidine buffer. NaCl and L-arginine hydrochloride excipient levels were explored at three levels: 10, 80, and 150 mM, as these were desired as potential stabilizers, viscosity reducers, and osmotic regulators. L-sorbitol excipient levels were selected at 50, 150, and 250 mM, as these were desired as potential stabilizers, cryoprotectants, and osmotic regulators. Poloxamer 188 was explored at 0.005, 0.0275, and 0.05% (w / v), as this was desired as a surfactant to prevent protein aggregation and particle formation. Total antibody concentration levels were validated at 100, 125, and 150 mg / mL to provide sufficient concentrations for subcutaneous administration of the formulation. Using JMP statistical software, a definitive design of experiment (DOE) was created to obtain 18 formulations using seven factors, each with three levels. This DOE was applied to eptinezumab-LuAG00922 co-formulation ratios of 1:1, 1:2, and 2:1 to obtain a total of 54 formulations.

[0346] Formulation composition and buffer exchange Table 3 below shows the reagents used in the preparation of the formulations. Standard stock solutions for each formulation component were prepared as follows: 0.20 M L-histidine, 1.0 M sodium chloride (NaCl), 3.5 M sorbitol, 0.9 M L-arginine hydrochloride, and 10.0% (w / v) poloxamer 188. The 18 formulation buffers shown in Table 4 were prepared by diluting an appropriate amount of each standard stock solution for each formulation component with distilled deionized water (Milli-Q water). The final pH of each formulation buffer was adjusted with 5 M hydrochloric acid and filtered through a 0.22 μm PES filter.

[0347] [Table 5]

[0348] Eighteen formulation buffers containing L-histidine, L-sorbitol, and L-arginine hydrochloride were mixed with appropriate amounts of standard stock solution, and then diluted with distilled deionized water (Milli-Q water) to obtain the compositions shown in the table below. Three ratios of eptinezumab to LuAG00922 were selected (1:2, 1:1, and 2:1), and each was used to exchange buffer in the 18 formulations to produce a total of 54 protein formulations using Big Tuna (Unchained Labs).

[0349] [Table 6]

[0350] The stability of the 54 formulations was evaluated by size exclusion column using ultra-high pressure liquid chromatography under the following conditions and time points. 5℃ (0, 1, 4, 7 months) 25℃ (1, 4, 7 months) 40℃ (1 month)

[0351] method Protein concentration measurement: After buffer exchange with the desired co-formulation, protein concentrations were measured using Solo VPE and an Agilent Cary 60 UV-Vis spectrophotometer or Lunatic spectrophotometer (Unchained Labs), along with known A280 extinction coefficients for eptinezumab and LuAG00922.

[0352] High-throughput assay: Size exclusion chromatography was performed along with ultra-high pressure liquid chromatography (SE-UPLC) using a Waters Acquity H-class UPLC equipped with a TUV detector connected to a Waters BEH SEC column (P / N 186005225). SE-UPLC analysis was performed at T=0 and at 5°C, 25°C, 30°C, and 40°C to evaluate the percentage of high molecular weight (HMW%) species. The initial HMW% of eptinezumab was 0.32%, and the initial HMW% of LuAG00922 was 0.94%. For each of the 54 co-formulations after buffer exchange, the total HMW% result at T=0 ranged from 0.39% to 1.40%. JMP DOE model analysis revealed significant effects on the HMW% response from changes in protein concentration, eptinezumab to LuAG00922 ratio, histidine concentration, L-arginine hydrochloride concentration, pH, and sorbitol concentration. Sodium chloride concentration and poloxamer (P188) concentration did not show any significant changes in the HMW% response. Uncle (Unchained Labs) conducted high-throughput viscosity measurements using a bead method based on dynamic light scattering (DLS) (He et al. 2010, Analytical Biochemistry, Volume 399, Issue 1, Pages 141-143, High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions). Polystyrene beads with a diameter of 100 nm (ThermoScientific: P / N 3100A) were added to the protein formulation.

[0353] [Table 7]

[0354] [Table 8]

[0355] [Table 9]

[0356] Example 8 The following data was obtained in the same manner as in Example 7, but the stability test was performed at 30°C, and SE-UPLC analysis was performed at T=10 days and T=20 days.

[0357] [Table 10]

[0358] Example 9 The following data was obtained in the same manner as in Example 7, but the stability test was performed at 30°C, and SE-UPLC analysis was performed at T=10 days and T=20 days.

[0359] [Table 11]

[0360] [Table 12]

[0361] [Table 13]

Claims

1. A pharmaceutical composition comprising eptinezumab and LuAG09222, wherein eptinezumab comprises the VH region described in SEQ ID NO: 4 and the VL region described in SEQ ID NO: 10, and LuAG09222 comprises the VH region described in SEQ ID NO: 15 and the VL region described in SEQ ID NO:

20.

2. The pharmaceutical composition according to claim 1, formulated to maintain the biological activity and / or storage stability of the eptinezumab and LuAG09222 antibody contained therein.

3. The pharmaceutical composition according to claim 1 or 2, which maintains the biological activity and / or storage stability of the eptinezumab and LuAG09222 antibody contained therein for at least one month, at least two months, at least three months, at least three to six months, at least six to nine months, at least nine to twelve months, or at least one year.

4. A pharmaceutical composition according to any one of the prior claims, comprising or further comprising histidine and either polysorbate 80 or poloxamer 188.

5. A pharmaceutical composition according to any one of the prior claims, comprising one, two, or all of the following excipients: NaCl, sorbitol, and arginine, or further comprising the above.

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

7. A pharmaceutical composition according to any one of the prior claims, comprising eptinezumab and LuAG09222 in a total concentration of approximately 100 mg / mL, approximately 150 mg / mL, approximately 200 mg / mL, approximately 250 mg / mL, or approximately 300 mg / mL.

8. The pharmaceutical composition according to claim 6 or 7, comprising an eptinezumab to LuAG09222 ratio of approximately 1:1, 1:2, or 2:

2.

9. A pharmaceutical composition according to any one of the prior claims, comprising approximately 100 to 300 mg / mL of eptinezumab and approximately 50 to 100 mg / mL of LuAG09222.

10. A pharmaceutical composition according to any one of the prior claims, comprising approximately 100 mg / mL of eptinezumab and approximately 50 mg / mL of LuAG09222.

11. The pharmaceutical composition according to any one of the prior claims, wherein eptinezumab and LuAG0922 are the sole active ingredients in the composition.

12. The pharmaceutical composition according to any one of the prior claims, wherein histidine is concentrated in a concentration of 10 to 50 mM, optionally in the range of about 20 to 40 mM, poloxamer P188 is concentrated in a concentration of 0.0025 to 0.0120% w / v, and polysorbate 80 is concentrated in a concentration of 0.005 to 0.05% w / v (including both extreme values).

13. A pharmaceutical composition according to any one of the prior claims, wherein the concentration of NaCl is approximately 10 to 150 mM, optionally approximately 30 to 70 mM; the concentration of sorbitol (optionally L-sorbitol) is 50 to 250 mM, optionally approximately 90 to 180 mM; and the concentration of arginine (optionally L-arginine) is 50 to 250 mM (including both extreme values).

14. A pharmaceutical composition according to any one of the prior claims, wherein the pH is approximately 5.0 to 6.8 (including both extreme values).

15. A pharmaceutical composition according to any one of the prior claims, wherein the pH is approximately 5.0, approximately 5.5, approximately 5.9, approximately 6.0, approximately 6.5, or approximately 6.

8.

16. A pharmaceutical composition according to any one of the prior claims, which is suitable for intravenous or subcutaneous administration.

17. A pharmaceutical composition according to any one of the prior claims, comprising eptinezumab and LuAG90222 in a ratio of about 2:1, about 1:1, or about 1:2, and comprising about 20 to 40 mM histidine buffer, about 90 to 180 mM sorbitol, 0.0025 to 0.0120% w / v poloxamer P188, and 30 to 70 mM NaCl, and having a pH of about 6, optionally about 5.

9.

18. A pharmaceutical composition according to any one of the prior claims, comprising eptinezumab and LuAG90222 in a 2:1 ratio, 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 5.

9.

19. A pharmaceutical composition according to any one of the prior claims, comprising eptinezumab and LuAG90222 in a 1:1 ratio, 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. A pharmaceutical composition according to any one of the prior claims, comprising eptinezumab and LuAG90222 in a 1:2 ratio, 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 prior claims, for use as a pharmaceutical.

22. A pharmaceutical composition according to any one of the prior claims, for use in the treatment or prevention of headache, optionally chronic or recurrent migraine or cluster headache, wherein administration of the pharmaceutical composition comprising the combination of antibodies has an additive or synergistic effect on inhibiting, reducing or preventing the frequency, duration and / or severity of migraine episodes compared to subcutaneous or intravenous administration of the same dose of eptinezumab or LuAG09222 alone.

23. A pharmaceutical composition according to any one of the prior claims, for use in the treatment or prevention of pain, optionally acute pain, chronic pain, neuropathic pain, nociceptive pain and / or radiculopathy, further optionally, the administration of the pharmaceutical composition comprising the combination of antibodies has an additive or synergistic effect on inhibiting or reducing pain compared to subcutaneous or intravenous administration of the same dose of eptinezumab or LuAG09222 alone.

24. A pharmaceutical composition according to any one of the prior claims, for administration monthly (every four weeks) or every two weeks.

25. The pharmaceutical composition according to any one of the prior claims, comprising eptinezumab, a heavy chain described in SEQ ID NO: 5 or SEQ ID NO: 6, and a light chain described in SEQ ID NO:

11.

26. LUAG09222 is a pharmaceutical composition according to any one of the prior claims, comprising a heavy chain described in SEQ ID NO: 16 or SEQ ID NO: 26 and a light chain described in SEQ ID NO:

21.

27. A method for treating or preventing headache, optionally chronic or recurrent migraine or cluster headache, comprising or comprising subcutaneous or intravenous administration of a combination of eptinezumab and the LuAG09222 antibody, wherein eptinezumab comprises the VH region described in SEQ ID NO: 4 and the VL region described in SEQ ID NO: 10, and LuAG09222 comprises the VH region described in SEQ ID NO: 15 and the VL region described in SEQ ID NO: 20, wherein optionally, the administration of the antibody combination has an additive or synergistic effect on inhibiting, reducing or preventing the frequency, duration and / or intensity of migraine episodes compared to subcutaneous or intravenous administration of the same dose of eptinezumab or LuAG09222 alone.

28. A method for treating or preventing pain, optionally acute pain, chronic pain, neuropathic pain, nociceptive pain and / or radiculopathy, comprising or comprising subcutaneous or intravenous administration of a combination of eptinezumab and LuAG09222 antibody, wherein eptinezumab comprises the VH region described in SEQ ID NO: 4 and the VL region described in SEQ ID NO: 10, and LuAG09222 comprises the VH region described in SEQ ID NO: 15 and the VL region described in SEQ ID NO: 20, wherein optionally, the administration of the antibody combination has an additive or synergistic effect on inhibiting or reducing pain compared to subcutaneous or intravenous administration of the same dose of eptinezumab or LuAG09222 alone.

29. The method according to claim 27 or 28, wherein the eptinezumab and LuAG09222 antibody are administered subcutaneously or intravenously by administration of the pharmaceutical composition according to any one of claims 1 to 20.

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